JOURNAL TRANSCRIPT
Datum: Sonntag, 01.03.2015 13:45 15:15 15:15 15:45 15:45 17:15 17:15 18:00 18:00 19:00 19:00 22:30
Workshop Ionisation unter Atmosphärendruck Teil 1
Workshop Lipidomics und Lipidanalytik Teil 1
Ort: I28 Chair: Hendrik Kersten
Ort: I27 Chair: Robert Ahrends, Dominik Schwudke
Kaffeepause Ort: Foyer
Workshop Ionisation unter Atmosphärendruck Teil 2
Workshop Lipidomics und Lipidanalytik Teil 2
Ort: I28 Chair: Hendrik Kersten
Ort: I27 Chair: Robert Ahrends, Dominik Schwudke
Tagungseröffnung Ort: K33
Wolfgang-Paul-Vorlesung Ort: K33
Welcome Reception Ort: Foyer
Präsentationen Wolfgang-Paul-Vorlesung Zeit: Sonntag, 01.03.2015: 18:00 - 19:00 · Ort: K33
Midwinter Reflections on the Progress of Biological Mass Spectrometry Catherine E. Costello Boston University School of Medicine, USA; cecmsms@bu.edu
Datum: Montag, 02.03.2015 8:30 9:15 9:15 10:15 10:15 10:45 10:45 11:10 11:10 12:50 12:50 14:20 14:20 15:05 15:05 15:50 15:50 16:05 16:05 17:25
Plenarvortrag I: Ionization at atmospheric pressure: why and how
17:25 17:40 17:40 18:40 18:40 20:25
Kaffeepause
Ort: K33
OIa: Bildgebende Massenspektrometrie OIb: Umwelt- & Lebensmittelanalytik I I Ort: K32 Ort: K33
OIc: Grundlagen der Massenspektrometrie Ort: I26
Agilent Research Summer Ort: K33
Kaffeepause Ort: Foyer
OIIa: Proteomics I
OIIb: Metabolomics
OIIc: Instrumentelle Entwicklungen
Ort: K33
Ort: K32
Ort: I26
Mittagspause / Lunchseminare
Plenarvortrag II: How to speed up high-performance mass spectrometry Ort: K33
Wolfgang-Paul-Studienpreis Ort: K33
Kaffeepause Ort: Foyer
OIIIa: Ionisationsmethoden I
OIIIb: Lipide & Phospholipide I
Ort: K33
Ort: K32
OIIIc: Gasphasenreaktionen, Fragmentierungen & Strukturaufklärung Ort: I26
Ort: Foyer
Gastvortrag: Der Weg wissenschaftlicher Erkenntnisse in die Politik - oder wie wird Wissenschaft wirksam? Ort: K33
PI: Postersession I Ort: Foyer
Präsentationen Plenarvortrag I Zeit: Montag, 02.03.2015: 8:30 - 9:15 · Ort: K33
Ionization at atmospheric pressure: why and how Andries Bruins University of Groningen, Niederlande; a.p.bruins@rug.nl
OIa: Bildgebende Massenspektrometrie I Zeit: Montag, 02.03.2015: 9:15 - 10:15 · Ort: K33
OIa1: MALDI-2: Sensitive MS Imaging with Laser-Induced Postionization at 5 Micrometer Pixel Size Jens Soltwisch1, Hans Kettling1,2, Simeon Vens-Cappel1,2, Marcel Wiegelmann1, Johannes Müthing1,2, Klaus Dreisewerd1,2 1 Institute for Hygiene, University of Münster, Germany; 2Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Germany; dreisew@uni-muenster.de
OIa2: Spatially resolved detection of contact and systemic pesticides in plants using DESI-MSI Stefanie Gerbig1, Hubertus Brunn2, Bernhard Spengler1, Sabine Schulz1 1 2 Justus Liebig Universität Gießen, Deutschland; Landesbetrieb stefanie.gerbig@anorg.chemie.uni-giessen.de
Hessisches
Landeslabor,
Gießen,
Deutschland;
OIa3: Mass spectrometry imaging for characterizing parasite host interactions in malaria. Saleh Mahmud Khalil1, Andreas Römpp1, Jette Pretzel2, Katja Becker2, Bernhard Spengler1 1 Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany; 2Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, Germany; saleh0207@gmail.com
OIb: Umwelt- & Lebensmittelanalytik I Zeit: Montag, 02.03.2015: 9:15 - 10:15 · Ort: K32
OIb1: Analyse von Fruchtsäften mittels HPLC/CZE-IMS-qTOF-MS Oliver Schmitz, Susanne Stephan Universität Duisburg-Essen, Angewandte Analytische Chemie; oliver.schmitz@uni-due.de
OIb2: A non-targeted LC-HRMS multi-method for the detection of food allergen trace contaminations Robin Korte, Jens Brockmeyer Westfälische Wilhelms-Universität Münster, Deutschland; r.korte@posteo.de
OIb3: DART-FT-ICR-MS-Studie zur Abgabe von Polydimethylsiloxanen in Lebensmittel beim Gebrauch von SilicongummiHaushaltsartikeln Jürgen Gross Universität Heidelberg, Deutschland; juergen.gross@oci.uni-heidelberg.de
OIc: Grundlagen der Massenspektrometrie Zeit: Montag, 02.03.2015: 9:15 - 10:15 · Ort: I26
OIc1: Studying viral assemblies with mass spectrometry and XFELs Charlotte Uetrecht Heinrich Pette Institut, Deutschland; lottiu@yahoo.de
OIc2: UV-MALDI-MS Analysis of Non-Covalent Streptavidin-Biotin Complexes with a 6-Aza-2-thiothymine Matrix: Effect of Wavelength and Fluence on their Detection Andreas Schnapp1, Marcel Wiegelmann1, Jens Soltwisch1, Klaus Dreisewerd1,2 1 Institute for Hygiene, University of Münster, Germany; 2Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Germany; mail@andreas-schnapp.de
OIc3: Fragmentierung von Ethylaminosubstituierten Farbstoffen mittels CID und PD Claus Gernert, Jürgen Grotemeyer Christian Albrechts Universität zu Kiel, Deutschland; gernert@phc.uni-kiel.de
Agilent Research Summer Zeit: Montag, 02.03.2015: 10:15 - 10:45 · Ort: K33
N-Maleoyl Amino Acids as Novel Alkylating Agents for Mass-Spectrometric Detection of Cysteine-Containing Peptides – a Complex Comparison for Complex Proteomics Wiebke Nadler1,2,3, Regina Berg4, Philipp Walch2,3, Mathis Baalmann3, Christoph Rösli1,2 1 Deutsches Krebsforschungszentrum, Deutschland; 2Heidelberg Institute for Stem Cell Technology (HI-STEM); Heidelberg; 4Department of Organic Chemistry, University of Basel; wiebke.nadler@hi-stem.de
3
Ruprecht-Karls-University
OIIa: Proteomics I Zeit: Montag, 02.03.2015: 11:10 - 12:50 · Ort: K33
OIIa1: Towards a mass spectrometric Western Blot: Multiplexed Targeted Protein Quantification by Gel-LC-MS/MS Marc Gentzel, Mukesh Kumar, David Drechsel, Andrej Shevchenko MPI of Molecular Cell Biology and Genetics, Dresden, Deutschland; gentzel@mpi-cbg.de
OIIa2: A universal workflow for efficient enrichment of proteins in mass-spectrometry based proteomics Florian Bonn, Jürgen Bartel, Knut Büttner, Michael Hecker, Andreas Otto, Dörte Becher Institut für Mikrobiologie, Universität Greifswald, Deutschland; fbonn@uni-greifswald.de
OIIa3: Characterization of an Improved Ultra-High Resolution Quadrupole Time of Flight (UHR-QTOF) Instrument for Proteomics Applications Stephanie Kaspar, Markus Lubeck, Annette Michalski, Oliver Raether, Christoph Gebhardt, Stuart Pengelley Bruker Daltonik GmbH, Bemen, Deutschland; Stuart.Pengelley@bruker.com
OIIa4: Thermal Proteome Profiling MIkhail Savitski, Friedrich Reinhard, Gerard Drewes, Marcus Bantscheff Cellzome GmbH, Deutschland; marcus.x.bantscheff@gsk.com
OIIa5: Uncovering molecular details of protein "misfolding - aggregation" using affinity- and ion mobility- mass spectrometry: Physiological and Parkinson- Synucleins Michael Przybylski1, Kathrin Lindner1, Nicolas Pierson2, Ying Zhang3, Brindusa-Alina Petre1,3, Stefan Schildknecht1, Michael Gross3, David Clemmer2 1 Steinbeis Centzre for Biopolymer Analysis & Biomedical Mass Spectrometry, University of Konstanz, and Rüsselsheim, Germany; 2Department of Chemistry, Indiana University, Bloomington, USA; 3Department of Chemistry, Washington University St.Louis, USA; michael.przybylski@stw.de
OIIb: Metabolomics Zeit: Montag, 02.03.2015: 11:10 - 12:50 · Ort: K32
OIIb1: Negative Ion Electrospray Tandem Mass Spectrometry of Polyketides Jürgen Schmidt Leibniz Institute of Plant Biochemistry Halle (Saale), Germany; jschmidt@ipb-halle.de
OIIb2: Combination of proteomic and metabolomic approaches in plant stress research Andrej Frolov1,2, Elena Lukasheva3, Tatiana Bilova1,2, Gagan Paudel1,2, Uta Greifenhagen1,2, Dominic Brauch1,2, Elena Tarakhovskaya3, Juliane Mittasch4, Natalia Osmolovskaja3, Gerd Ulrich Balcke5, Carsten Milkowski4, Claudia Birkemeyer6, Ludger Wessjohann7, Ralf Hoffmann1,2 1 Universität Leipzig, Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry; 2Center for Biotechnology and Biomedicine (BBZ), Leipzig; 3Saint-Petersburg State University, Faculty of Biology, Department of Plant Biochemistry and Physiology; 4Martin Luther University HalleWittenberg, Interdisciplinary Center for Crop Plant Research (IZN); 5Institute of Plant Biochemistry, Department of Cell and Metabolic Biology; 6 Universität Leipzig, Faculty of Chemistry and Minerology, Institute of Analytical Chemistry; 7Institute of Plant Biochemistry, Department of Bioorganic Chemistry; andrej.frolov@bbz.uni-leipzig.de
OIIb3: Online EC/(LC)/ESI-MS for investigating the oxidative metabolism of roxarsone Lisa Frensemeier, Lars Büter, Uwe Karst University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 48149 Münster, Deutschland; lisa.frensemeier@unimuenster.de
OIIb4: Metabolic pathway driven targeted metabolomics – a “quickstep” from mass spectrometric raw data to biologically relevant conclusions Anrdea Kiehne, Aiko Barsch, Verena Tellström, Heiko Neuweger Bruker Daltonik GmbH, Bremen, Deutschland; Verena.Tellstroem@bruker.com
OIIc: Instrumentelle Entwicklungen Zeit: Montag, 02.03.2015: 11:10 - 12:50 · Ort: I26
OIIc1: Improving resolving power in complex mixtures by combining tunable synchrotron radiation with advanced mass spectrometric techniques Arnas Lucassen Physikalisch-Technische Bundesanstalt, Deutschland; arnas.lucassen@ptb.de
OIIc2: Hochempfindliches Fourier-Transform-Massenspektrometer mit nicht-destruktiver Ionendetektion Michael Schmidt1, Albrecht Brockhaus1, Stefan Butzmann1, Thorsten Benter2, Alexander Laue3, Michel Aliman3 1 Institut für Sensorik und messtechnische Systeme, Bergische Universität Wuppertal; 2Institut für Physikalische und Theoretische Chemie, Bergische Universität Wuppertal; 3Carl Zeiss SMT GmbH; mschmidt@uni-wuppertal.de
OIIc3: Coupling thin layer chromatography with mass spectrometry - a practical approach for matrix-loaded samples Hans Griesinger1, Katerina Matheis1, Jürgen Schiller3, Beate Fuchs3, Michael Schulz2 1 Merck KGaA, Department of Bioanalytical Chemistry, Deutschland; 2Merck KGaA, Instrumental Analytics R&D, Deutschland; 3Universität Leipzig, Institut für Medizinische Physik und Biophysik, Deutschland; hans.griesinger@merckgroup.com
OIIc4: Proton-Transfer-Reaction Mass Spectrometry Coupled with FastGC: High Selectivity in Near Real-Time Philipp Sulzer1, Rene Gutmann1, Lukas Fischer1,2, Jens Herbig1, Simone Jürschik1, Alfons Jordan1, Eugen Hartungen1, Gernot Hanel1, Lukas Märk1, Matteo Lanza1,2, Kostiantyn Breiev1,2, Tilmann D. Märk1,2 1 IONICON Analytik GmbH., Eduard-Bodem-Gasse 3, 6020 Innsbruck, AUSTRIA; 2Institut für Ionenphysik und Angewandte Physik, LeopoldFranzens Universität Innsbruck, Technikerstr. 25, 6020 Innsbruck, AUSTRIA; philipp.sulzer@ionicon.com
OIIc5: RECENT ADVANCES IN TIME-OF-FLIGHT MASS SPECTROMETRY: WHAT CAN MULTIPLEXING DO FOR YOU? Gerhard Horner2, Laura McGregor3, Nick BUKOWSKI3, Leonhard Pollack1, Stefan Koschinski1 1 Markes International GmbH, Deutschland; 2five technologies GmbH, Deutschland; 3Markes International Ltd., UK; ghorner@markes.com
Plenarvortrag II Zeit: Montag, 02.03.2015: 14:20 - 15:05 · Ort: K33
How to speed up high-performance mass spectrometry Yury O. Tsybin1,2, Konstantin O. Nagornov1, Kristina Srzentic1, Anton N. Kozhinov1 1 École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; 2Spectroswiss Sàrl, 1015 Lausanne, Switzerland; yury.tsybin@epfl.ch
Wolfgang-Paul-Studienpreis Zeit: Montag, 02.03.2015: 15:05 - 15:50 · Ort: K33
Charakterisierung und Optimierung von Corona - Mikroplasma initiierten Ionisationsprozessen zur Anwendung in der Atmosphärendruckionisations - Massenspektrometrie Sonja Klee Tofwerk AG, Schweiz; klee@tofwerk.com
Mechanistische Studien zur Mizoroki-Heckmassenspektrometrischer Methoden
und
zur
Kobalt(I)-katalysierten
Lukas Fiebig Boehringer Ingelheim Pharma GmbH & Co. KG, Deutschland; lukas.fiebig@boehringer-ingelheim.com
Diels-Alder-Reaktion
mithilfe
OIIIa: Ionisationsmethoden I Zeit: Montag, 02.03.2015: 16:05 - 17:25 · Ort: K33
OIIIa1: Highly time-resolved two-dimensional mass spectrometric imaging of molecular combustion and pyrolysis product concentrations in a burning cigarette Ralf Zimmermann1, Thorsten Streibel1, Romy Hertz-Schünemann1, Kevin McAdam2, Chuan Liu2, Sven Ehlert1 1 Uni Rostock, Deutschland; 2BAT R&D, UK; ralf.zimmermann@gsf.de
OIIIa2: Laserspektroskopische Untersuchung an heterosubstituierten Halogenbenzolen mittels REMPI- und MATISpektroskopie Sascha Krüger, Jürgen Grotemeyer Christian-Albrechts-Universität zu Kiel, Deutschland; krueger@phc.uni-kiel.de
OIIIa3: Matrixeffekte bei der LC/MS-Analyse von Vitamin D-Metaboliten mittels verschiedener Ionisierungstechniken Sebastian Hagenhoff, Heiko Hayen Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Deutschland; sebastian.hagenhoff@unimuenster.de
OIIIa4: Three-layer set-up for standardized quantification of salicylic acid in dried blood spots by desorption electrospray ionization (DESI) mass spectrometry Markus Siebenhaar1,2,3, Kai Küllmer3, Nuno Miguel de Barros Fernandes3, Volker Hüllen3, Carsten Hopf1,2 1 Instrumental Analysis and Bioanalysis, Department of Biotechnology, Mannheim University of Applied Sciences; 2Institute of Medical Technology, University of Heidelberg and Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim, Germany; 3Roche Diagnostics GmbH, Sandhofer Straße 116, 68305 Mannheim; Markus.Siebenhaar@roche.com
OIIIb: Lipide & Phospholipide I Zeit: Montag, 02.03.2015: 16:05 - 17:25 · Ort: K32
OIIIb1: LMP-EX a multimolecular omics approach for systems biology Robert Ahrends, Cristina Coman ISAS, Deutschland; Ahrends@gmx.net
OIIIb2: Analysis of the lipid composition of adipose tissues by spectroscopic methods Yulia Popkova1, Andrej Meusel1, Jana Breitfeld2, Dorit Schleinitz2, Johannes Hirrlinger3,4, Dirk Dannenberger5, Peter Kovacs2, Jürgen Schiller1 1 University of Leipzig, Medical Faculty, Institute of Medical Physics and Biophysics, Härtelstrasse 16- 18, 04107 Leipzig, Germany; 2University of Leipzig, IFB Adiposity Diseases, Liebigstrasse 21, 04103 Leipzig, Germany; 3University of Leipzig, Medical Faculty, Carl-Ludwig-Institute for Physiology, Liebigstrasse 27, 04103 Leipzig, Germany; 4Dept. of Neurogenetics, Max-Planck-Institut for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany; 5Leibniz Institute for Farm Animal Biology, Institute of Muscle Biology and Growth, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany; yulia.popkova@medizin.uni-leipzig.de
OIIIb3: Gender-specific Differences in Blood Plasma Lipidomes of Healthy Females and Males Susanne Sales1, Juergen Graessler2, Marc Bickle1, Carlo Cannistraci3, Kim Ekroos4, Andrej Shevchenko1 1 Max Planck Institute of Molecular Cell Biology and Genetics, Germany; 2Department of Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany; 3Biotechnology Center TU Dresden, Germany; 4Zora Biosciences Oy, Espoo, Finnland; sales@mpi-cbg.de
OIIIb4: Lipidomics screens of human lung tissue together with histological characterization allow studying lipid metabolic perturbation in cancer and COPD Lars F. Eggers1, Julia Müller2, Verena Scholz1, Torsten Goldmann2,3, Dominik Schwudke1,3 1 Division of Bioanalytical Chemistry - Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany; 2Division of Clinical and Experimental Pathology - Research Center Borstel, Parkallee 1-40, 23845 Borstel, Germany; 3Airway Research Center North – German Center for Lung Research, Wöhrendamm 80, 22927 Grosshansdorf, Germany; leggers@fz-borstel.de
OIIIc: Gasphasenreaktionen, Fragmentierungen & Strukturaufklärung Zeit: Montag, 02.03.2015: 16:05 - 17:25 · Ort: I26
OIIIc1: A new TEMPO-Active Ester Reagent for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) Mass Spectrometry Mathias Schäfer1, Christian Ihling2, Francesco Falvo1, Isabel Kratochvil2, Andrea Sinz2 1 Department of Chemistry, University Cologne, Greinstrasse 4, 50939 Köln, Germany; 2Institute of Pharmacy, Martin-Luther University HalleWittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany.; mathias.schaefer@uni-koeln.de
OIIIc2: First Principles Calculation of Electron Ionization Mass Spectra Christoph Bauer, Stefan Grimme Universität Bonn, Deutschland; christoph.bauer@thch.uni-bonn.de
OIIIc3: Structure of Full-Length p53 Tumor Suppressor Probed by Chemical Cross-Linking and Mass Spectrometry Christian Arlt, Christian Ihling, Andrea Sinz Institute of Pharmaceutical Chemistry & Bioanalytics, Martin-Luther University Halle-Wittenberg; christian.arlt@pharmazie.uni-halle.de
OIIIc4: Identification, recognition structures and affinities of nitrated tyrosine sites by affinity mass spectrometry Claudia Andries1,2, Stefan Slamnoiou1, Laura Ion1,2, Michael Przybylski1, Brindusa-Alina Petre1,2 1 Steinbeis Centre for Biopolymer Analysis & Biomedical Mass Spectrometry, Konstanz & Rüsselsheim, Germany; 2Faculty of Chemistry, A.I.Cuza University of Iasi, Romania; brindusa.petre@uaic.ro
Gastvortrag Zeit: Montag, 02.03.2015: 17:40 - 18:40 · Ort: K33
Der Weg wissenschaftlicher Erkenntnisse in die Politik - oder wie wird Wissenschaft wirksam? Uwe Schneidewind Wuppertal Institut für Klima, Umwelt, Energie, Deutschland; uwe.schneidewind@wupperinst.org
PI: Postersession I Zeit: Montag, 02.03.2015: 18:40 - 20:25 · Ort: Foyer
Analytische Anwendungen PI01: Combining thin-layer chromatography, antibody-overlay detection and DESI-FT-ICR-MS for the analysis of Shiga toxin glycosphingolipid receptors Katja Steckhan1, Christoph Große Kracht1, Lothar Veith2, Carsten Engelhard3, Daniel Steil1, Iris Meisen1, Johannes Müthing1, Michael Mormann1 1 Institute for Hygiene, University of Münster, Robert-Koch-Straße 41, 48149 Münster, Germany; 2Tascon GmbH, Mendelstraße 17, 48149 Münster, Germany; 3Institute for Analytical Chemistry, University of Siegen, Adolf-Reichwein-Straße 2, 57068 Siegen, Germany; k_stec01@uni-muenster.de
PI02: Massenspektrometrische Analyse von Biotensiden am Beispiel der Rhamnolipide Beate Behrens1, Jeannine Engelen2, Heiko Hayen1 1 Westfälische Wilhelms-Universität Münster, Deutschland; 2Bergische Universität Wuppertal, Deutschland; behrensb@uni-muenster.de
PI03: Bestimmung von polaren Metaboliten in Algenextrakten mittels HILIC-ESI-MS Alexander Schriewer, Heiko Hayen Westfälische Wilhelms-Universität Münster, Deutschland; alexander.schriewer@wwu.de
PI04: Bringing Triple Quadrupole Detection Limits to a New Standard –in Theory and in Practice Ralf Falter, Thomas Glauner, Laszlo Toelgyesi Agilent Technologie, Deutschland; ralf_falter@agilent.com
PI05: Identification of urinary peptides triggering social behavior of Danio Rerio by mass spectrometry Astrid Markgraf1, Jens Christoffers2, Gabriele Gerlach1 1 Institute of Biology and Environmental Sciences, Carl von Ossietzky University, D- 26111 Oldenburg, Germany; 2Institute of Chemistry and Center of Interface Science, Carl von Ossietzky University, D-26111 Oldenburg, Germany; astrid.markgraf@uni-oldenburg.de
PI06: Advancements in (MA)LDI based mass spectrometry imaging techniques in the field of chemical ecology Filip Kaftan1, Jerrit Weißflog1, Samay Pande2, Purva Kulkarni3, Mayuri Napagoda4, Tino Jaschinski5, Aleš Svatoš1 1 Max Planck Institute for Chemical Ecology, Deutschland; 2Institute of Integrative Biology, Department of Environmental Systems, Zürich, Switzerland; 3Chair for Bioinformatics, Friedrich Schiller University Jena, Jena, Germany; 4Faculty of Medicine, University of Ruhuna, Department of Biochemistry, Sri Lanka; 5Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Germany; fkaftan@ice.mpg.de
Bildgebende Massenspektrometrie PI07: Determination of the platinum distribution in Caenorhabditis elegans after Cisplatin incubation by LA-ICP-MS Barbara Crone1, Julia Bornhorst2, Michael Aschner3, Tanja Schwerdtle2, Uwe Karst1 1 Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany; 2Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany; 3Department of Molecular Pharmacology, Neuroscience, and Pediatrics, Albert Einstein College of Medicine, Bronx NY, USA; barbaracrone@wwu.de
PI08: Exploring the head and neck cancer by MALDI FT-ICR mass spectrometry imaging Lukas Krasny1, Franziska Hoffmann2, Günther Ernst2, Jan Hendrik Kobarg3, Dennis Trede3,4, Theodore Alexandrov4,5,6, Vladimir Havlicek1, Orlando Guntinas-Lichius7, Ferdinand von Eggeling2,7,8, Anna C. Crecelius9,10 1 Institute of Microbiology, v.v.i., Prague, Czech Republic; 2Institute of Physical Chemistry, Friedrich Schiller University Jena, Jena, Germany; 3 Steinbeis Innovation Center SCiLS Research, Bremen, Germany; 4SCiLS GmbH, Bremen, Germany; 5Center for Industrial Mathematics, University of Bremen, Bremen, Germany; 6European Molecular Biology Laboratory, Heidelberg, Germany; 7Department of Otorhinolaryngology, Jena University Hospital, Jena, Germany; 8Leibniz Institute of Photonic Technology (IPHT), Jena, Germany; 9Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Jena, Germany; 10Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Jena, Germany; kobarg@scils.de
PI09: Laser ablation dielectric barrier discharge imaging MS for direct molecular analysis of pharmaceuticals in tablets Tim Elseberg1, Christina Herdering1, Michael Sperling2, Uwe Karst1 1 University of Münster, Institute of Inorganic and Analytical Chemistry; tim.elseberg@uni-muenster.de
2
European Virtual Institute for Speciation Analysis (EVISA);
PI10: Improved spatial resolution in the analysis of FFPE tissue after tryptic digestion Janine Beckmann, Detlev Suckau, Michael Becker, Arndt Asperger Bruker Daltonik GmbH, Bremen, Deutschland; Arndt.Asperger@bruker.com
PI11: Untersuchung der Verteilung von Silbernanopartikeln nach oraler Gabe im Gastrointestinaltrakt von Ratten mit LAICP-MS Dörthe Dietrich1, Dr. Antje Vennemann2, Prof. Dr. Martin Wiemann2, Prof. Dr. Uwe Karst1 1 Westfälische Wilhelms-Universität Münster, Deutschland; 2IBE R&D gGmbH; d_diet03@uni-muenster.de
PI12: Quantitative Bioimaging of Pd-tagged Photosensitizers in Tumor Spheroids by LA-ICP-MS Ann-Christin Niehoff1,2, Rebecca Niehaus2, Michael Sperling2, Uwe Karst2 1 Graduate School of Chemistry, University of Muenster; 2Institute of Inorganic and Analytical Chemistry, University of Muenster; a.niehoff@unimuenster.de
PI13: AP-SMALDI mass spectrometry imaging of metabolites in insects using high resolution in mass and space Dhaka Bhandari, Matthias Schott, Andreas Römpp, Andreas Vilcinskas, Bernhard Spengler Justus Liebig University Giessen, Deutschland; Dhaka.R.Bhandari@anorg.Chemie.uni-giessen.de
PI14: Differentiating Macrophages in atherosclerotic plaques using matrix-assisted laser desorption/ionization mass spectrometry imaging Pegah Khamehgir-Silz1, Andreas H. Wagner2, Andreas Römpp1, Markus Hecker2, Bernhard Spengler1 1 Institute of Inorganic and Analytical Chemistry, Justus Liebig University, Germany; 2Institute of Physiology and Pathophysiology, University of Heidelberg, Germany; Pegah.Khamehgir@anorg.chemie.uni-giessen.de
Datenenalyse PI15: Customized data processing pipeline based on command line tools Henrik Thomas, Andrej Shevchenko MPI-CBG, Deutschland; thomas@mpi-cbg.de
Element- & Speziationsanalytik PI16: LC-ICP-MS and LC-ESI-MS for the Speciation Analysis of Iodine Maria Viehoff1, Chun Kong Mak1, Michael Sperling2, Uwe Karst1 1 University of Muenster, Institute of Inorganic and Analytical Chemistry, Germany; 2European Virtual Institute for Speciation Analysis (EVISA), Muenster, Germany; mariaviehoff@uni-muenster.de
Gasphasenreraktionen, Fragmentierungen & Strukturaufklärung PI17: Resonanzverstärkte Multiphotonenionisation von Triethylbenzolen Heinke Thurn, Jürgen Grotemeyer Christian Albrechts Universität zu Kiel, Deutschland; thurn@phc.uni-kiel.de
Grundlagen der Massenspektrometrie PI18: Photofragmentierung von Azofarbstoffen im sichtbaren Wellenlängenbereich Martin Clemen, Jürgen Grotemeyer Christian-Albrechts-Universität zu Kiel, Deutschland; clemen@phc.uni-kiel.de
PI19: Revisiting Classical MALDI Matrices: The Effect of Laser Wavelength Annika Koch1, Andreas Schnapp1, Marcel Wiegelmann1, Jens Soltwisch1, Klaus Dreisewerd1,2 1 University of Münster, Institute of Hygiene, Biomedical Mass Spectrometry, Robert-Koch-Str. 41, 48149 Münster, Germany; 2Interdisciplinary Center for Clinical Research (IZKF) Münster; Annika.Koch@uni-muenster.de
PI20: Influence of transfer capillary temperature on adduct formation in AP-MALDI MS Anna Schultheis, Bernhard Spengler Justus Liebig University Giessen, Germany; anna.schultheis@anorg.chemie.uni-giessen.de
Hyphenated Techniques PI21: Sensitivity of detection of 12 aminoglycoside antibiotics by positive ESI-MS - comparison between HILIC and reverse phase methods Jens Boertz1, Olga Shimelis2, Emily Barrey2, Dave Bell2, Craig Aurand2 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Sigma-Aldrich, Supelco, Bellefonte, USA; jens.boertz@sial.com
PI22: Rapid LC-MS/MS determination of dioxin and digitoxin in biological fluids with minimal matrix effects Jens Boertz1, Xiaoning Lu2, Hillel Brandes2, Stacy Squillario2, David Bell2, Wayne Way2 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Sigma-Aldrich, Supelco, Bellefonte, USA; jens.boertz@sial.com
PI23: Determination of thyroid hormones in biological fluids by LC-MS with online solid phase extraction Jens Boertz1, Xiaoning Lu2, Dave Bell2 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Sigma-Aldrich, Supelco, Bellefonte, USA; jens.boertz@sial.com
PI24: Thermogravimetry hyphenated to ultra-high resolution FT-ICR-mass spectrometry using atmospheric pressure chemical ionization (APCI) as novel approach for evolved gas analysis Toni Miersch1, Christopher P. Rüger1, Martin Sklorz1, Ralf Zimmermann1,2 1 Joint Mass Spectrometry Centre / Chair of Analytical Chemistry, University of Rostock, Rostock, Germany; 2Cooperation Group Comprehensive Molecular Analysis, Helmholtz Zentrum München, Neuherberg, Germany; toni.miersch@uni-rostock.de
Instrumentelle Entwicklungen PI25: Near-IR laser induced desorption sampling of acoustically levitated liquids Carsten Warschat1, Arne Stindt1, Andreas Bierstedt1, Ulrich Panne1,2, Jens Riedel1 1 BAM Federal Institute for Materials Research and Testing, Berlin, Germany; Carsten.Warschat@bam.de
2
HUB Humboldt University of Berlin, Germany;
PI26: Ein Orbitrap-Prototyp zur hochauflösenden Untersuchung von Erdöl Alessandro Vetere, Wolfgang Schrader Max-Planck-Inst. für Kohlenforschung, Deutschland; vetere@kofo.mpg.de
PI27: Assessing the Peptide Quantitation Performance of a Newly Developed Triple Quadrupole Instrument Ralf Falter, Volker Gnau, Christine Miller Agilent Technologie, Deutschland; ralf_falter@agilent.com
PI28: Eine DBD zur in-situ Erzeugung von Reaktantionen für negative chemische Ionisation Sascha Albrecht1, Fred Stroh1, Thorsten Benter2 1 Forschungszentrum Jülich GmbH, Deutschland; 2Bergische Universität Wuppertal, Deutschland; s.albrecht@fz-juelich.de
Life Sciences & Forensik PI29: Increasing Depth of Coverage in Data Independent Acquisition Joerg Dojahn1, Christie Hunter2, Ben Collins3, Ludovic Gillet3, Rudi Aebersold3 1 AB SCIEX, Deutschland; 2AB SCIEX, USA; 3ETH Zurich, Zurich, SWITZERLAND; joerg.dojahn@absciex.com
PI30: A targeted lipidomics approach for ceramide analysis in platelet Bing Peng, Cristina Coman, Robert Ahrends Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Deutschland; bing.peng@isas.de
PI31: Metabolic profiling of the battle field of competing white-rot fungi Riya C Menezes1, Marco Kai2, Aleš Svatoš1 1 Max Planck Institute for Chemical Ecology, Jena, Deutschland; 2Universität Rostock, Rostock, Deutschland; rmenezes@ice.mpg.de
PI32: Online Photostability Study of Pharmaceutical Substances Diego Zulkiewicz Gomes1,2, Jaber Assaf1, Thomas Schulze1, Maria Kristina Parr1 1 Freie Universität Berlin, Deutschland; 2IPT – Institute for Technological Research, Brazil; diegozg@zedat.fu-berlin.de
Lipide & Phospholipide PI33: To which extent are oxidized phospholipids digestible by the enzyme phospholipase A2? - A mass spectrometric and NMR spectroscopic study Jenny Schröter, Zschörnig Kristin, Fuchs Beate, Süß Rosemarie, Schiller Jürgen University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics; Jenny.Schroeter@medizin.uni-leipzig.de
PI34: δipidomics of Alzheimer’s Disease using an Integrated εicrofluidic-Ion Mobility-MS Device Gunnar Weibchen1, Steven Lai2, David Heywood2, Angela Doneanu2, Jim Murphy2, James Langridge3, Giuseppe Astarita2 1 Waters GmbH, Deutschland; 2Waters Corporation, Milford, MA, USA; 3Waters Corporation, Manchester, UK; gunnar_weibchen@waters.com
Metabolomics PI35: The Use of Fragment Ion and Collision Cross Section for Confident Identification from LC-Ion Mobility-MS Metabolomics Data Gunnar Weibchen1, Giorgis Isaac2, Giuseppe Astarita2, Steven Lai2, Adam Ladak2, James Langridge4, John Shockcor2, Andy Borthwick3 1 Waters GmbH, Deutschland; 2Waters Corporation, Milford, MA; 3Nonlinear Dynamics, Newcastle, UK; 4Waters Corporation, Manchester, UK; gunnar_weibchen@waters.com
PI36: Unravelling 2-deoxy-2-fluoro-D-glucose metabolism in plant tissue using mass spectrometry and NMR Amol Fatangare1, Christian Paetz2, Hans Peter Saluz3, Ales Svatos1 1 Mass spectrometry and proteomics research group, Max Planck institute for chemical ecology, Jena, Germany; 2Biosynthesis / NMR research group, Max Planck institute for chemical ecology, Jena, Germany; 3Cell and molecular biology department, Hans Knoll institute, Jena, Germany.; afatangare@ice.mpg.de
PI37: Synthese und massenspektrometrische Charakterisierung von Aib-haltigen antibiotischen Peptiden Annegret Laub Leibniz-Institut für Pflanzenbiochemie, Deutschland; Annegret.Laub@ipb-halle.de
Native MS & non-covalent Interactions PI38: Advances in Native Mass Spectrometry-based methods for the analysis of Non-Covalent Protein complexes Jonathan P. Williams Williams2, Marc Kipping1, Malcolm Anderson2, Lidia Jackson2, Kevin Giles2, Jeff Brown2 1 Waters GmbH, Deutschland; 2Waters Corporation, Manchester, UNITED KINGDOM; marc_kipping@waters.com
PI39: Bacteriophage endolysin activity is modulated by quaternary state. Boris Krichel1, Matt Dunne2, Rob Meijers2, Charlotte Uetrecht1 1 Heinrich Pette Institut, Deutschland; 2European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, DE-69117 Heidelberg, Germany.; boris.krichel@hpi.uni-hamburg.de
Petrolomics, Energy & Combustion PI40: Thermal/optical Carbon Analysis coupled with Photoionization Time-of-Flight Mass Spectrometry: Fine Particulate Matter from a Marine Engine Hendryk Czech1, Olli Sippula2,3, Martin Sklorz1, Thorsten Streibel1,3, Ralf Zimmermann1,3 1 Joint Mass Spectrometry Centre University of Rostock, Chair of Analytical Chemistry, Germany and Helmholtz-Zentrum München, CMA (Comprehensive Molecular Analytics), Germany; 2University of Eastern Finland, Department of Environmental Science, Fine Particle and Aerosol Technology; 3Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health (HICE) - www.hice-vi.eu; hendryk.czech@unirostock.de
Cross-Linking PI41: Characterization of the PKD2-ARF1 Complex by Chemical Cross-Linking and Mass Spectrometry Björn Häupl, Dirk Tänzler, Christian Ihling, Andrea Sinz Martin-Luther-Universität Halle-Wittenberg, Deutschland; bjoern.haeupl@pharmazie.uni-halle.de
PI44: Characterization of the Interaction between Intrinsically Disordered Gab1 and Grb2 by Cross-Linking/Mass Spectrometry Nadja Mallock1, Dirk Tänzler1, Katharina Mandel2, Marc Lewitzky2, Stephan Feller2, Andrea Sinz1 1 Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Germany; 2Tumor Biology Section, Institute of Molecular Medicine, Martin-Luther-University Halle-Wittenberg, Germany; nadja.mallock@pharmazie.uni-halle.de
PI51: Automated Assignment of MS/MS Cleavable Cross-Links in Protein 3D-Structure Analysis Michael Götze1, Jens Pettelkau2, Romy Fritzsche2, Christian Ihling2, Mathias Schäfer3, Andrea Sinz2 1 Institut für Biochemie und Biotechnologie, MLU Halle, Deutschland; 2Institut für Pharmazie, MLU Halle, Deutschland; 3Institut für Organische Chemie, Universität Köln, Deutschland; christian.ihling@pharmazie.uni-halle.de
Proteomics PI42: SpheriCal® - Monodisperse Polyester Dendrimers as Universal Mass Calibrants in Mass Spectrometry Jens Boertz1, Scott M. Grayson2, Michael Malkoch3 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Tulane University, Department of Chemistry, New Orleans, USA; 3Polymer Factory Sweden AB, Stockholm, Sweden; jens.boertz@sial.com
PI43: SILAC-Based Secretome Analysis of Non-Small Cell Lung Cancer Cell Lines Konstanze Bosse1, Silvia Haneder2, Christian Ihling1, Thomas Seufferlein3, Andrea Sinz1 1 Martin-Luther-Universität Halle, Deutschland; 2Spherotec GmbH Martinsried, Deutschland; konstanze.bosse@pharmazie.uni-halle.de
3
Universitätsklinikum
Ulm,
Deutschland;
PI45: Human bone marrow stromal cell-derived osteoblast matrix vesicle proteome and functions are regulated by sulfated glycosaminoglycan derivatives Johannes Schmidt1, Stefanie Kliemt1,2, Carolin Preissler3, Stephanie Möller4, Martin von Bergen1,5, Ute Hempel3, Stefan Kalkhof1 1 Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Germany; 2B CUBE Center for Molecular Bioengineering, TU Dresden, Germany; 3 Institute of Physiological Chemistry, TU Dresden, Germany; 4Biomaterials Department, INNOVENT e.V., Jena, Germany; 5Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark; johannes.schmidt@ufz.de
PI46: Monitoring PPAR induced changes in glycolysis by selected reaction monitoring mass spectrometry Andreas Hentschel, Cristina Coman, Robert Ahrends Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V.; andreas.Hentschel@isas.de
PI47: Proteomic study of the impact of Magnesium implants on osteoblasts Maryam Omidi1, Anna Burmester2, Parnian Kiani1, Marcel Kwiatkowski1, Berengere Luthringer2, Regine Willumeit2, Hartmut Schlüter1 1 Department of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Helmholtz-Zentrum Geesthacht; Institute of Materials Research; Structural Research on Macromolecules; Geesthacht, Germany; momidi@uke.de
PI48: Uncontrolled modification of redox-active cysteines in proteins and peptides Malte Bayer, Simone König Core Unit Proteomics, Interdisciplinary Center for Clinical Research, University of Münster; maltebayer@web.de
PI49: Low abundant N-linked glycosylation in wild-type hen egg white lysozyme is localized at non-consensus sequons Arndt Asperger1, Kristina Marx1, Christian Albers1, Laura Molin2, Odra Pinato2 1 Bruker Daltonik GmbH, Bremen, Deutschland; 2Chelab Silliker, Resana, Italy; arndt.asperger@bruker.com
PI50: A closer look at the quality of microwave digests Vera Jüngst, Marion Bäumlisberger, Ute Bahr, Michael Karas Goethe Universität Frankfurt, Deutschland; juengst@pharmchem.uni-frankfurt.de
PI52: New Workflows for Identification and Profiling of Disulfide Bonds in Biopharmaceuticals Jan Wiesner, Antje Kozicki, Anja Resemann, Rainer Paape, Lars Vorwerg, Kristina Marx, Andrea Kiehne, Ralf Hartmer, Carsten Baessmann, Detlev Suckau, Wolfgang Jabs, Zoltan Centnar Bruker Daltonik GmbH, Deutschland; Zoltan.Centnar@bruker.com
PI53: Exploring the potential of the last generation UHR-Q-TOF for rapid generation of accurate information on proteoforms distribution and relative abundancy Wolfgang Jabs, Stuart Pengelley, Christian Albers, P.O Schmit P.O Schmit, Verena Tellström Bruker Daltonik GmbH, Deutschland; Verena.Tellstroem@bruker.com
PI54: High quantification efficiency for discovery and validation approaches on a Q-TOF platform Stephanie Kaspar1, Pierre-Olivier Schmit2, Ulrike Schweiger-Hufnagel1, Aiko Barsch1, Stuart Pengelley1 1 Bruker Daltonik GmbH, Bremen, Deutschland; 2Bruker Daltonique S.A., Wissembourg, France; Stuart.Pengelley@bruker.com
PI55: MeCAT - Using light for cleavability David Benda, Sabine Ufer, Sebastian Beck, Michael W. Linscheid Humboldt-Universität zu Berlin, Deutschland; david.benda.1@chemie.hu-berlin.de
PI56: Displacement chromatography as enrichment step in phosphoproteomics Parnian Kiani, Marcel Kwiatkowski, Hartmut Prof. Dr. Schlüter Mass Spectrometric Proteomics, Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf (UKE); p.kiani@uke.de
PI57: Mass spectrometric based approaches for studying oligomerization of -amyloid peptide at amino acids level Andries Claudia1, Ion Laura1, Michael Gross2, Petre Brindusa Alina1,2 1 Alexandru Ioan Cuza University, Iasi, Romania, Rumänien; 2Washington University, St. Louis, Missouri, USA; andries.claudia17@gmail.com
PI58: A top to bottom approach for in-depth characterization of therapeutic monoclonal antibodies Martin Samonig1,2, Kai Scheffler1,3, Christian Huber1,4 1 Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization; 2Thermo Fisher Scientific, Dornierstraße 4, 82110 Germering, Germany; 3Thermo Fisher Scientific GmbH, Im Steingrund 4-6, 63303 Dreieich, Germany; 4University of Salzburg, Department of Molecular Biology, Division of Chemistry and Bioanalytics, Hellbrunnerstrasse 34, 5020 Salzburg, Austria; kai.scheffler@thermofisher.com
PI59: Targeted quantification of myofibrillar myopathy aggregate related proteins via PRM Katalin Barkovits1, Alexandra Maerkens1,2, Rudi A. Kley2, Matthias Vorgerd2, Katrin Marcus3 1 Department of Medical Bioanalytics, Medizinisches Proteom-Center, Ruhr-University Bochum; 2Department of Neurology, Neuromuscular Centre Ruhrgebiet, University Hospital Bergmannsheil, Ruhr-University Bochum; 3Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-University Bochum; katalin.barkovits@rub.de
PI65: Identifizierung von intrazellulären Cisplatin-Protein Addukten mithilfe des fluoreszierenden Cisplatin-Analogon CFDA-Cisplatin Sandra Kotz1, Maximilian Kullmann2, Anya Kalayda2, Ulrich Jaehde2, Sabine Metzger1 1 Universität zu Köln, Deutschland; 2Universität Bonn, Deutschland; skotz@uni-koeln.de
Umwelt- & Lebensmittelanalytik PI60: UPLC Ion Mobility Mass Spectrometry: A New Approach to Authentication and Routine Screening of Ginsenocide Isomers in Functional Food Products. Michael McCullagh1, Davor Turkovic2, Rob Lewis1, David Douce1 1 Waters Corporation, UK; 2Waters GmbH, Deutschland; davor_turkovic@waters.com
PI61: The Combining of an Integrated Microfluidic Device with CCS Ion Mobility Screening for the Analysis of Pesticide Residues in Food. Michael McCullagh1, Davor Turkovic2, Christoph Thomas2, David Douce1 1 Waters Corp., UK; 2Waters GmbH, Deutschland; christoph_thomas@waters.com
PI62: Screening and Quantitation of About 350 Pesticides in Fruit Juices with Positive/Negative Switching LC/MS/MS Zicheng Yang1, Louis Maljers1, Arnd Ingendoh2 1 Bruker Daltonics Inc., USA; 2Bruker Daltonik GmbH, Deutschland; Arnd.Ingendoh@bruker.com
PI63: Screening und Quantifizierung abwasserbürtiger Spurenstoffe in einer Uferfiltrationstransekte mittels UHPLC-HRMS Patricia van Baar, Florian Wode, Uwe Dünnbier Berliner Wasserbetriebe, Deutschland; patricia.vanbaar@bwb.de
PI64: A DESI MS based screening method for phthalates in consumer goods Sabine Schulz1, Sebastian Wagner1, Stefanie Gerbig1, Dieter Bohn2, Detlef Sielaff3, Herbert Wächter4, Bernhard Spengler1 1 Justus-Liebig-Universität, Deutschland; 2Landesbetrieb Hessisches Landeslabor, Deutschland; 3Landesuntersuchungsamt Rheinland-Pfalz, Deutschland; 4Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Deutschland; sabine.schulz@anorg.chemie.uni-giessen.de
Datum: Dienstag, 03.03.2015 8:30 9:15 9:15 10:15 10:15 10:45 10:45 11:10 11:10 12:50 12:50 14:20 14:20 15:05 15:05 15:50 15:50 17:35 17:35 18:35 19:30 23:00
Plenarvortrag III: Merging mass spectrometry and IR spectroscopy Ort: K33
OIVa: Proteomics II
OIVb: Bildgebende Massenspektrometrie II
OIVc: Hochauflösende Massenspektrometrie
Ort: K32
Ort: I26
OVa: Cross-Linking
OVb: Lipide & Phospholipide II
OVc: Ionenmobilität
Ort: K33
Ort: K32
Ort: I26
Ort: K33
Mattauch-Herzog-Förderpreis Ort: K33
Kaffeepause Ort: Foyer
Mittagspause / Lunchseminare
Plenarvortrag IV: Human Proteomes: From basic science to understanding drug action Ort: K33
Preis Massenspektrometrie in den Biowissenschaften Ort: K33
PII: Postersession II Ort: Foyer
Mitgliederversammlung Ort: K33
Konferenzdinner Ort: Brauhaus
Wuppertal
Präsentationen Plenarvortrag III Zeit: Dienstag, 03.03.2015: 8:30 - 9:15 · Ort: K33
Merging mass spectrometry and IR spectroscopy Jos Oomens1,2 1 Radboud University Nijmegen, Institute for Molecules and Materials, Niederlande; 2Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, Niederlande; j.oomens@science.ru.nl
OIVa: Proteomics II Zeit: Dienstag, 03.03.2015: 9:15 - 10:15 · Ort: K33
OIVa1: Age-related changes of advanced glycation patterns in Arabidopsis thaliana Tatiana Bilova1,2,3, Dominic Brauch1,2, Uta Greifenhagen1,2, Elena Tarakhovskaya3, Claudia Birkemeyer4, Ludger Wessjohann5, Andrej Frolov1,2 1 Institut fuer Bioanalytische Chemie, Fakutltaet fuer Chemie und Mineralogie, Universitaet Leipzig; 2Biomedizinisch-Biotechnologisches Zentrum (BBZ), Universitaet Leipzig; 3Biological Faculty of Saint-Petersburg State University, Russia; 4Institut fuer Analytische Chemie, Fakultaet fuer Chemie und Mineralogie, Universitaet Leipzig; 5Natur- und Wirkstoffchemie, Leibniz-Institut fuer Pflanzenbiochemie; bilova.tatiana@gmail.com
OIVa1: Probing pH-Dependent Protein G´e Surface Topology Alterations by Fast Photochemical Oxidation of Proteins and Mass Spectrometry Yelena Yefremova1, Mahmoud Al-Majdoub1, Kwabena F.M. Opuni1, Cornelia Koy1, Yuetian Yan2, Michael Gross2, Michael O. Glocker1 1 Proteome Center Rostock, University Medicine Rostock, Rostock, Germany; 2Washington University in St. Louis, St. Louis, Missouri, USA; yelena.yefremova@uni-rostock.de
OIVa3: Oxidative degradation of N -fructosylamine-substituted peptides in heated aqueous systems Uta Greifenhagen, Viktor Sekovski, Marko Damjanovic, Andrej Frolov, Ralf Hoffmann Institut für Bioanalytische Chemie, Fakultät für Chemie und Mineralogie; Biotechnologisch-Biomedizinisches Zentrum, Universität Leipzig, Deutschland; uta.greifenhagen@bbz.uni-leipzig.de
OIVb: Bildgebende Massenspektrometrie II Zeit: Dienstag, 03.03.2015: 9:15 - 10:15 · Ort: K32
OIVb1: Mass Spectrometry Imaging of Biological Tissue: An Approach for Multicenter Studies Andreas Römpp1, Jean-Pierre Both2, Alain Brunelle3, Ron M. A. Heeren4, Olivier Laprevote5, Brendan Prideaux6, Alexandre Seyer3, Markus Stoeckli7, Donald F. Smith8, Bernhard Spengler1 1 Justus-Liebig-Universität Giessen, Deutschland; 2French Atomic Energy Commission (CEA-LIST), Saclay, France; 3Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France; 4The Maastricht Multimodal Molecular Imaging institute (M4I), Maastricht University, Maastricht, The Netherlands; 5 Chimie Toxicologie Analytique et Cellulaire, Université Paris Descartes, Paris, France; 6Public Health Research Institute, UMDNJ, Newark, NJ, United States; 7Novartis Institutes for BioMedical Research, Basel, Switzerland; 8National High Magnetic Field Laboratory, Florida State University, Tallahassee Florida, United States; Andreas.Roempp@anorg.Chemie.uni-giessen.de
OIVb2: Genaueste Einblicke in die Klimageschichte anhand der Bestimmung von Lipidbiomarkern in marinen Sedimenten mittels LDI FTICR Imaging Lars Wörmer1, Susanne Alfken1, Marcus Elvert1, Jens Fuchser2, Julius S. Lipp1, Matthias Zabel3, Kai-Uwe Hinrichs1 1 Organic Geochemistry Group, MARUM Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, Germany; 2Bruker Daltonik GmbH, Deutschland; 3Inorganic Geochemistry Group, MARUM Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, Germany; Jens.Fuchser@bruker.com
OIVb3: Combining label-free whole-cell MALDI MS biotyping and imaging mass spectrometry for in-situ monitoring of histone deacetylase drug target activation Bogdan Munteanu1,2, Björn Meyer1,2, Elke Burgermeister3, Carolina v. Reitzenstein1,2, Matthias P. Ebert3, Carsten Hopf1,2 1 Center for Applied Research in Biomedical Mass Spectrometry (ABIMAS); 2Instrumentelle Analytik und Bioanalytik Hochschule Mannheim; 3 Universitätsklinikum Medizinische Fakultät Mannheim der Universität Heidelberg; munteanu@hs-mannheim.de
OIVc: Hochauflösende Massenspektrometrie Zeit: Dienstag, 03.03.2015: 9:15 - 10:15 · Ort: I26
OIVc1: High precision mass measurement and separation of nuclear isomers with a multiple-reflection time-of-flight mass spectrometer Christine Hornung1, Samuel Ayet1,2, Timo Dickel1,2, Jens Ebert1, Hans Geissel1,2, Emma Haettner2, Ronja Knöbel2, Ivan Miskun2, Stephane Pietri2, Wolfgang R. Plaß1,2, Sivaji Purushothaman2, Moritz Pascal Reiter1, Ann-Kathrin Rink1, Helmut Weick2, Peter Dendooven3, Marcel Diwisch1, Florian Greiner1, Fabian Heiße2, Christian Jesch1, Nasser Kalanta-Nayestanaki2, Johannes Lang1, Wayne Lippert1, Iain Moore4, Alexandre Pikhtelev5, Ilkka Pohjalaine4, Andrej Prochazka2, Manisha Ranjan3, Christoph Scheidenberger1,2, Maya Takechi2, John S. Winfeld2, Xiaodong Xu1,2, Mikhail I. Yavor6 1 JLU Gießen, Germany; 2GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany; 3KVI, University of Groningen, 9747 AA Groningen, The Netherlands; 4University of Jyväskylä, FI-40014 Jyväskylä, Finland; 5Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Russia; 6Institute for Analytical Instrumentation, Russian Academy of Sciences, 190103 St. Petersburg, Russia; christine.hornung@physik.uni-giessen.de
OIVc2: Analysis of polar trace components in middle distillate fuel by ultra-high resolution mass spectrometry using ESI and GC-APCI Christopher Paul Rüger1, Elize Smit2, Martin Sklorz1, Stefan De Goede3, Ralf Zimmermann1,4, Egmont Rohwer2 1 Joint Mass Spectrometry Centre / Chair of Analytical Chemistry, University of Rostock, Rostock, Germany; 2Department of Chemistry, University of Pretoria, Pretoriam South Africa; 3Sasol Technology, Fuels Research, Sasol, Sasolburg, South Africa; 4Cooperation Group Comprehensive Molecular Analysis, Helmholtz Zentrum München, Neuherberg, Germany; christopher.rueger@uni-rostock.de
OIVc3: Adding a chemical dimension to the analysis of heavy crude oils with ultra-high resolution mass spectrometry Xuxiao Wang, Wolfgang Schrader Max-Planck-Institut für Kohlenforschung; wxuxiao@kofo.mpg.de
Mattauch-Herzog-Förderpreis Zeit: Dienstag, 03.03.2015: 10:15 - 10:45 · Ort: K33
ESI Mass Spectrometry for Tracking Down Elusive Organometallics Konrad Koszinowski Georg-August-Universität Göttingen, Deutschland; kkoszin@gwdg.de
OVa: Cross-Linking Zeit: Dienstag, 03.03.2015: 11:10 - 12:50 · Ort: K33
OVa1: Improving cross-linked peptide identification in large protein complexes across different high-resolution LC/MS/MS platforms Christof Lenz1,2, Chung-Tien Lee1,2, Olexandr Dybkov1, Reinhard Lührmann1, Henning Urlaub1,2 1 Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Deutschland; 2Universitätsmedizin christof.lenz@mpibpc.mpg.de
OVa2: Monitoring Conformational Changes in PPAR / Spectrometry
Göttingen
(UMG),
Deutschland;
by Chemical Cross-Linking, Photo-Affinity Labeling, and Mass
Rico Schwarz, Dirk Tänzler, Knut Kölbel, Christian Ihling, Andrea Sinz MLU Halle-Wittenberg, Deutschland; rico.schwarz@pharmazie.uni-halle.de
OVa3: Chemical crosslinking of TSHR-ECD Marcus M. B. Nagel1,2, Jörg Schaarschmidt1, Jens Meiler3, Stefan Kalkhof2, Ralf Paschke1 1 University of Leipzig, Division of Endocrinology and Nephrology; 2Helmholtz Centre for Environmental Research – UFZ, Department of Proteomics, Leipzig, Germany; 3Vanderbilt University, Center for Structural Biology, Nashville, USA; marcus.nagel@ufz.de
OVa4: Incorporation of Photo-Methionine into Calmodulin for Photo-Cross-Linking/MS Studies Christine Piotrowski, Christian Ihling, Andrea Sinz Department of Pharmaceutical Chemistry & Bioanalytics, christine.piotrowski@pharmazie.uni-halle.de
Institute
of
Pharmacy,
OVa5: Mass spectrometric analysis of post-translational modifications in T cells Christian Freund Freie Universität Berlin, Deutschland; christian.freund@fu-berlin.de
Martin-Luther
University
Halle-Wittenberg;
OVb: Lipide & Phospholipide II Zeit: Dienstag, 03.03.2015: 11:10 - 12:50 · Ort: K32
OVb1: Lipids originating from Mycobacterium tuberculosis (Mtb) are detected in plasma of Tuberculosis Patients Nicole Zehethofer1,2, Jan Heyckendorf3, Ulrich E. Schaible1, Christoph Lange3, Dominik Schwudke2 1 Research Center Borstel, Cellular Microbiology, Parkallee 1-40, Borstel, Germany;; 2Research Center Borstel, Bioanalytical Chemistry, Parkallee 1-40, Borstel, Germany; 3Research Center Borstel, Clinical Infectious Diseases, Parkallee 1-40, Borstel, Germany; NZehethofer@fz-borstel.de
OVb2: A simple MALDI MS-based method to identify ether lipids in complex lipid mixtures of spermatozoa Beate Fuchs1, Karin Müller2, Jürgen Schiller1 1 University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Härtelstr. 16-18, D-04107 Leipzig; 2Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, D-10315 Berlin; beate.fuchs@medizin.uni-leipzig.de
OVb3: LPPdb: a new MS-centered database of modified lipids Zhixu Ni1,2, Ivana Milic1,2, Dieter Weber1,2, Andrea Annibal1,2, Ralf Hoffmann1,2, Maria Fedorova1,2 1 Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig,; 2Center for Biotechnology and Biomedicine, Universität Leipzig.; zhixu.ni@uni-leipzig.de
OVb4: Shotgun Lipidomics for Dissecting Lipid-protein Assemblies Sophie Ayciriex1, Hermeto Gerber2, Patrick Fraering2, Andrej Shevchenko1 1 Max Planck Institute of Molecular Cell Biology and Genetics, Deutschland; 2Ecole Polytechnique Fédérale de Lausanne (EPFL), Brain Mind Institute and School of Life Sciences, Switzerland; ayciriex@mpi-cbg.de
OVb5: LC-MS2 based Lipid Mediator Profiling reveals insights into signaling cascades during Mycobacterium tuberculosis infection Matthias Krajewski1, Bhesh Paudyal2, Ulrich Schaible2, Dominik Schwudke1 1 Forschungszentrum Borstel, Deutschland, Bioanalytische Chemie; 2Forschungszentrum Borstel, Deutschland, Zelluläre Mikrobiologie; mkrajewski@fz-borstel.de
OVc: Ionenmobilität Zeit: Dienstag, 03.03.2015: 11:10 - 12:50 · Ort: I26
OVc1: Eliminating systematic errors in the LC-MS/MS analysis of vitamin D in human serum Dietrich Volmer, Yulin Qi Universität des Saarlandes, Deutschland; Dietrich.Volmer@mx.uni-saarland.de
OVc2: The new Agilent IMS QTOF, how it works and where it does add advantages Thiemann Joachim, Ralf Falter Agilent Technologie, Deutschland; joachim_thiemann@agilent.com
OVc3: Weiterführende Untersuchungen Ionenmobilitätsspektrometrie
zum
Vergleich
von
APLI
und
APCI
bei
der
Anwendung
in
der
Marvin Ihlenborg, Björn Raupers, Jürgen Grotemeyer Christian-Albrechts-Universität zu Kiel, Deutschland; ihlenborg@phc.uni-kiel.de
OVc4: Gas-Phase Microsolvation of Ubiquitin: Identification of Crown Ether Binding Sites Melanie Göth1, Xiao Jakob Schmitt2, Stephan Warnke2, Gert von Helden2, Kevin Pagel1,2 1 Freie Universität Berlin, Institute of Chemistry and Biochemistry, Takustraße 3, 14195 Berlin, Germany; 2Fritz Haber Institute of the Max Planck Society, Department of Molecular Physics, Faradayweg 4-6, 14195 Berlin, Germany; m.goeth@fu-berlin.de
OVc5: Analysis of isomeric lipids by high resolution ion mobility-mass spectrometry Michael Größl1, Stephan Graf1, Miroslav Lisa2, Michal Holcapek2, Julio Sampaio3, Bernhard Dick4, Bruno Vogt4, Richard Knochenmuss1 1 Tofwerk, Schweiz; 2University of Pardubice, Czech Republic; 3Lipotype, Germany; 4Bern University Hospital, Switzerland; groessl@tofwerk.com
Plenarvortrag IV Zeit: Dienstag, 03.03.2015: 14:20 - 15:05 · Ort: K33
Human Proteomes: From basic science to understanding drug action Mathias Wilhelm1,2, Judith Schlegl2, Hannes Hahne1, Mikhail Savitski3, Amin Moghaddas Gholami1, Susan Kläger1, Daniel Martinez Molina4, Pär Nordlund4, Marcus Bantscheff3, Gerard Drewes3, Bernhard Küster1 1 Technische Universität München, Germany; 2SAP, Germany; 3Cellzome, Germany; 4Karolinska Institute, Sweden; kuster@tum.de
Preis Massenspektrometrie in den Biowissenschaften Zeit: Dienstag, 03.03.2015: 15:05 - 15:50 · Ort: K33
Stable isotope labelling and mass spectrometric analysis of peptides allows tracking of isotopic flux in microbial communities Martin von Bergen1, Jana Seifert2 1 Helmholtz Zentrum für Umweltforschung, Deutschland; 2Universität Hohenheim; Martin.vonbergen@ufz.de
PII: Postersession II Zeit: Dienstag, 03.03.2015: 15:50 - 17:35 · Ort: Foyer
Analytische Anwendungen PII01: Veränderungen des organischen Bodenkohlenstoffs bei Waldbränden Diana Hofmann1, Bernhard Steffen1, Kai-Uwe Eckhardt2, Peter Leinweber2 1 Forschungszentrum Jülich, Deutschland; 2Bodenkunde der Universität Rostock; d.hofmann@fz-juelich.de
PII02: Detection of interfering substances of enzyme-based glucose measurement in complex matrices by Flowprobe™ micro extraction mass spectrometry Tanja Gaißmaier1, Makus Siebenhaar1,2,3, Vanya Todorova2, Carsten Hopf1,3, Volker Hüllen2 1 Instrumental Analysis and Bioanalysis, Department of Biotechnology, Mannheim University of Applied Sciences,; 2Roche Diagnostics GmbH, Sandhofer Straße 116, 68305 Mannheim; 3Institute of Medical Technology, University of Heidelberg and Mannheim University of Applied Sciences; tanja.gaissmaier@gmail.com
PII03: Improved Determination of Allergenic Fragrances in Detergents and Personal Care Products in Multiple Reaction Monitoring GC-MS/MS Gordon van’t Slot, Verena Tellström Bruker Daltonik GmbH, Deutschland; verena.tellstroem@bruker.com
PII04: Direct Analysis of Degradation Products in Lithium-ion Batteries Using Low-temperature Plasma Ambient Desorption/Ionization High-Resolution Mass Spectrometry Christopher Kuhlmann1, Inessa Pastushkina1, Sascha Nowak2, Carsten Engelhard1 1 Universität Siegen, Deutschland; 2MEET - Münster Electrochemical Energy Technology, Deutschland; christopher.kuhlmann@uni-muenster.de
PII05: Automated top-down mass spectrometry of hemoglobin for a clinical application Didia Coelho Graça1, Adelina E. Acosta-Martin1,2, Wolfgang Jabs3, Ralf Hartmer3, Lorella Clerici2, Markus Meyer3, Kaveh Samii4, Yury O. Tsybin5, Denis Hochstrasser1,2, Pierre Lescuyer1,2, Alexander Scherl1,2, Zoltan Centnar3 1 Department of Human Protein Sciences, Faculty of Medicine, Geneva University, Geneva, Switzerland; 2Department of Genetic and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland; 3Bruker Daltonik GmbH, Bremen, Germany; 4Division of Hematology, Geneva University Hospital, Geneva, Switzerland; 5Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; Zoltan.Centnar@bruker.com
PII06: Electrospray ionization of aromatic amino compounds Andreas Kiontke1, Andrej Frolov2, Ariana Oliveira1, Carolin Oswald1, Claudia Birkemeyer1 1 Institute of Analytical Chemistry, University of Leipzig; 2Center for Biotechnology and Biomedicine, University of Leipzig; andreaskiontke@unileipzig.de
Bildgebende Massenspektrometrie PII07: Multiple mass spectrometric imaging analyses from single tissue section with Desorption Electrospray Ionization (DESI) on a oa-TOF mass spectrometer Gunnar Weibchen1, Emrys Jones2, Emmanuelle Claude2 1 Waters GmbH, Deutschland; 2Waters Corporation, Manchester, UK; gunnar_weibchen@waters.com
PII08: Enhancement of weak analyte signals in human disease-related MALDI MS images by applying fast randomized denoising and compression algorithms Matthias Schwartz, Björn Meyer, Bernhard Wirnitzer, Carsten Hopf Hochschule Mannheim, Deutschland; m.schwartz@hs-mannheim.de
PII09: Imaging mass spectrometry (IMS) to discriminate breast from pancreatic cancer metastasis in FFPE tissues Rita Casadonte1, Mark Kriegsmann2, Friederike Zweynert3, Katrin Friedrich4, Gustavo Barreton4, Mike Otto1,3,5, Sören Deininger6, Rainer Paape6, Eckhard Belau6, Detlev Suckau6, Daniela Aust4, Christian Pilarsky4, Jörg Kriegsmann1,3,5, Arndt Asperger6 1 Proteopath, Trier, Germany; 2University of Heidelberg, Department of Pathology, Heidelberg, Germany; 3Institute of Molecular Pathology, Trier, Germany; 4University of Dresden, Department of Pathology, Dresden, Germany; 5Center for Histology, Cytology and Molecular Diagnostics Trier, Germany; 6Bruker Daltonik GmbH, Bremen, Deutschland; Arndt.Asperger@bruker.com
PII10: 3D MALDI Imaging of Mouse Heart after Myocardial Infarction Michael Becker1, Lena Hauberg-Lotte2, Judith Lotz3, Janina Oetjen2, Dennis Trede9, Michaela Aichler6, Wolfgang Dreher7, Moritz Wildgruber8, Klaus Steinhorst5, Jan Hendrik Kobarg5, Stefan Schiffler9, Stefan Heldmann3, Herbert Thiele3, Peter Maass4, Axel Walch6, Theodore Alexandrov4, Arndt Asperger1 1 Bruker Daltonik GmbH, GERMANY; 2MALDI Imaging Lab, University of Bremen, Bremen, GERMANY; 3Fraunhofer MEVIS Project Group Image Registration, Lübeck, GERMANY; 4Center for Industrial Mathematics, Bremen, GERMANY; 5Steinbeis Innovation Center SCiLS Research, Bremen, GERMANY; 6Research Unit Analytical Pathology, HMGU München, Oberschleissheim, GERMANY; 7University of Bremen, Bremen, GERMANY; 8Klinikum Rechts der Isar, TU München, München, GERMANY; 9SCiLS GmbH, Bremen, GERMANY; Arndt.Asperger@bruker.com
PII11: LA-ICP-MS to Study the Distribution of Copper in Wilson´s Disease Liver Samples Oliver Hachmöller1, Michaela Aichler2, Ann-Christin Niehoff1, Michael Sperling1, Axel Walch2, Uwe Karst1 1 Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany; 2Research Unit Analytical Pathology, Helmholtz Zentrum München, München, Germany; o.hachmoeller@uni-muenster.de
PII12: Improving ion–signal reproducibility in MALDI MSI using an internal standard Sarah Aboulmagd1, Diego Esteban-Fernández1, Boris Neumann2,4, Alberto Lázaro3, Alberto Tejedor3, Michael Linscheid1 1 Institut für Chemie, Humboldt-Universität zu Berlin, Deutschland; 2Proteome Factory, Magnusstraße 11, 12489 Berlin, Germany; 3Renal Physiopathology Laboratory, Department of Nephrology, IiSGM-Hospital General Universitario Gregorio Maran˜o´n, Universidad Complutense de Madrid, Madrid, Spain; 4Charité-Universitätmedizin Berlin, Institute of Pharmacology, Hessische Straße 3-4, 10115 Berlin, Germany; sarah.aboulmagd80@gmail.com
PII13: Pre-separation of ions in an atmospheric-pressure MALDI ion source based on differential mobility in a frequencyasymmetric alternating electrical field Christof Barth, Bernhard Spengler Justus-Liebig-Universität Giessen; christof.barth@chemie.uni-giessen.de
Datenanalyse PII14: Chromatographic resolved mass defect plots: A new analytical tool for the interpretation of high-resolution MS data. Thomas Gröger1,2, Benedikt Weggler1,2, Ralf Zimmermann2,1 1 Helmholtz Zentrum München – German Research Center for Environmental Health GmbH, Deutschland; thomas.groeger@helmholtz-muenchen.de
2
University of Rostock;
Element- & Speziationsanalytik PII15: Metal labelling of antibodies Oleksandra Kuzmich, Michael Linscheid Humboldt Universität zu Berlin, Deutschland; aleksandrakuzmich@gmail.com
Gasphasenreaktionen, Fragmentierungen & Strukturaufklärung PII16: Reactivity of Hydrated Monovalent First Row Transition Metal Ions M+(H2O)n, M = Cu and Zn toward C6H5Cl, C6H5Br, C6H5I and C3H7l Ina Herber, Martin Beyer Institut für Ionenphysik und Angewandte Physik, Universität Innsbruck, Österreich; Ina.Herber@uibk.ac.at
PII17: Untersuchung der Fragmentierungen von auf Cyclen basierenden Ligandensystemen mittels ESI-MS/MS und MS³ Markus Plaumann1,2, Dorit Kemken1, Thomas Dülcks1, Dieter Leibfritz1 1 Universität Bremen, Institut für Organische und Analytische Chemie, Deutschland; Biometrie und Medizinische Informatik, Deutschland; markus.plaumann@med.ovgu.de
2
Otto-von-Guericke Universität Magdeburg, Institut für
Grundlagen der Massenspektrometrie PII18: Kinetic Energy Release und Fragmentierungswege substituierter Aniline Sarah Seulen, Jürgen Grotemeyer Christian-Albrechts-Universität zu Kiel, Deutschland; seulen@phc.uni-kiel.de
PII19: The Extraction of Maximum Information from Individual Ion Arrivals And Its Application to Extending the Dynamic Range of IMS-oaTOF-MS Data Martin Green1, Darrell Williams1, Garry Scott1, Tony Gilbert1, Martin Palmer1, Nick Tomczyk1, Keith Richardson1, Mark Wrona2, Alexander Muck3, Mathias Hofmann3 1 Waters Corporation, Wilmslow, UK; 2Waters Corporation, Milford, MA, USA; 3Waters GmbH, Eschborn, GE; alex_muck@waters.com
Hochauflösende Massenspektrometrie PII20: Expediting Peptide Mapping with High Resolution LC-MS Kai Scheffler1, Tabiwang Arrey2, Eugen Damoc2, Maciej Bromirski2 1 Thermo Fisher Scientific GmbH, Dreieich, Germany; 2Thermo Fisher Scientific, Bremen, Germany; kai.scheffler@thermofisher.com
Hyphenated Techniques PII21: Analysis of multiple toxins by LC-MS/MS: In-depth analysis of column selectivity Jens Boertz1, Emily Barrey2, Olga Shimelis2 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Sigma-Aldrich, Supelco, Bellefonte, USA; jens.boertz@sial.com
PII22: Sensitive and fast analysis of aflatoxin M1 in milk at picogram levels using LC-MS/MS Jens Boertz1, Ken Espenscheid2, Emily R. Barrey2, Olga I. Shimelis2, Michael Ye2, Jim Brown2, Ed Mauney2 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Sigma-Aldrich, Supelco, Bellefonte, USA; jens.boertz@sial.com
PII23: TLC-MALDI-MS investigation of a multi-compound flu medication Michael Schulz2, Hans Griesinger1, Michaela Oberle2, Knut Behrend3, Katerina Matheis1 1 Merck KGaA, Department of Bioanalytical Chemistry, Deutschland; 2Merck KGaA, Instrumental Analytics R&D, Deutschland; 3Universität Hamburg, Institut für Lebensmittelchemie, Deutschland; michael.schulz@merckgroup.com
PII24: Influence of the silica gel layer thickness on the quality of TLC-MALDI mass spectra of lipids Hans Griesinger1, Katerina Matheis1, Beate Fuchs3, Jürgen Schiller3, Michael Schulz2 1 Merck KGaA, Department of Bioanalytical Chemistry, Deutschland; 2Merck KGaA, Instrumental Analytics R&D, Deutschland; 3Universität Leipzig, Institut für Medizinische Physik und Biophysik, Deutschland; hans.griesinger@merckgroup.com
Instrumentelle Entwicklungen PII25: Exploring Impact of Dynamic Accumulation for Improving MS/MS Quality of QqTOF Data Joerg Dojahn1, Nic Bloomfield2, Christie Hunter2, Sean Seymour2 1 AB SCIEX, Deutschland; 2AB SCIEX, USA; joerg.dojahn@absciex.com
PII26: Design and Development of a New, High Performance 20kV HED Detector with Improved Efficiency and Low Noise Characteristics Ralf Falter, Layne Howard, Micahel Ugarov Agilent technologie, Deutschland; ralf_falter@agilent.com
PII27: Coupling of Acoustically Levitated Droplets with Ion Mobility Spectrometry Aleksandra Michalik-Onichimowska1,2, Toralf Beitz2, Jens Riedel1, Ulrich Panne1,3, Hans-Gerd Löhmannsröben2 1 BAM Federal Institute for Materials Research and Testing, Deutschland; 2University of Potsdam, Deutschland; 3Humboldt-Universität zu Berlin, Deutschland; aleksandra.michalik@bam.de
Life Sciences & Forensik PII28: Identification of human milk derived proteolytic glycopeptides by use of ion mobility separation (IMS) and subsequent low energy collision-induced dissociation Gottfried Pohlentz, Michael Mormann, Julia Krägenbring, Klaus Dreisewerd, Johannes Müthing Institut für Hygiene, Universität Münster, Deutschland; pohlentz@uni-muenster.de
PII29: Enrichment of Glycoproteins from Human Breast Milk and Structural Characterization of N-Glycosylation Julia Krägenbring, Gottfried Pohlentz, Michael Mormann Institute for Hygiene, Westfälische Wilhelm-University Münster, Germany; j.kraegenbring@uni-muenster.de
PII30: Enhanced Confirmation Criteria for Reducing False Positive Rates (FPR) in Toxicology Screening using High Resolution QToF Accurate Mass Analysis Tony Drury, Stuart Pengelley Bruker Daltonik GmbH, Bremen, Deutschland; Stuart.Pengelley@bruker.com
PII31: Introducing ion mobility mass spectrometry in glycomics of Schindler’s disease Mirela Sarbu1, Jasna Peter-Katalinic2, David Clemmer3, Michael Przybylski4, Alina Zamfir1 1 West University of Timisoara, Romania; 2Institute for Medical Physics and Biophysics, University of Muenster; 3Department of Chemistry, Indiana University, Bloomington, USA;; 4Steinbeis Center Biopolymer Analysis and Biomedical Mass Spectrometry, Rüsselsheim, Germany;; mirela.sarbu86@yahoo.co.uk
Lipide & Phospholipide PII32: The maturation of sperm is accompanied by changes of the (phospho-)lipid composition – A MALDI-TOF MS Investigation of murine epididymal spermatozoa Ariane Nimptsch1, Susanne Pyttel2, Jan Böttger3, Ulrike Jakop4, Jürgen Schiller1 1 Universität Leipzig - Medizinische Fakultät, Institut für Medizinische Physik und Biophysik; 2Universitätsklinikum Leipzig - Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie; 3Universität Leipzig - Medizinische Fakultät, Institut für Biochemie; 4Leibniz-Institut für Zoo- und Wildtierforschung (IZW) im Forschungsverbund Berlin e.V.; ariane.nimptsch@medizin.uni-leipzig.de
PII33: Phospholipid-Profiling mittels HILIC-ESI-MS/MS Christian Vosse, Heiko Hayen Westfälische Wilhelms-Universität Münster, Deutschland; christianvosse@uni-muenster.de
Metabolomics PII34: Metabolomics Profiling using Atmospheric Pressure Gas Chromatography-MS Gunnar Weibchen1, Vladimir Shulaev3, Ghaste Manoj3,4, Steven Lai2, Carolina Salazar3, Nobuhiro Suzuki3, Janna Crossley3, Ron Mittler3, James Langridge2, Giuseppe Astarita2, Fulvio Mattivi4 1 Waters GmbH, Deutschland; 2Waters Corporation, Milford, MA; 3University of North Texas, Denton, TX; 4Fondazione Edmund Mach, San Michele all'Adige, Italy; gunnar_weibchen@waters.com
PII35: A novel high resolution MS/MS Human Metabolite Spectral Library enabling rapid and accurate metabolite identification in human metabolomics studies Zhendong Li2, Mingguo Xu2, Yiman Wu2, Chiao-Li Tseng2, Tao Huan2, Wei Han2, Jaspaul Tatlay2, Tran Tran2, Aiko Barsch1, Carsten Baessmann1, Liang Li2, Stuart Pengelley1 1 Bruker Daltonik GmbH, Bremen, Deutschland; 2University of Alberta, Edmonton, CANADA; Stuart.Pengelley@bruker.com
PII36: Quantifizierung von TMAO ernährungsphysiologischer Studien
und
verwandter
Metabolite
im
Plasma
mit
HILIC-MS/MS
im
Kontext
Ralf Krüger1, Maria Pfeuffer1, Michael Empl2, Pablo Steinberg2, Alexander Roth1, Achim Bub1 1 Max Rubner-Institut, Karlsruhe; 2Tierärztliche Hochschule Hannover; ralf.krueger@mri.bund.de
PII37: An extraction strategy for systems biology Cristina Coman, Fiorella Solari, Andreas Hentschel, René Zahedi, Robert Ahrends Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Deutschland; cristina.coman@isas.de
PII65: Lysosomal Storage Diseases: Development of ESI- ion trap diagnostic assay for MPS II and MPS VI. Multiplexing possibilities Laura Ion1,2, Claudia Andries1,2, Michael Przybylski1, Brindusa-Alina Petre1,2 1 Steinbeis Centre for Biopolymer Analysis, Univ. of Konstanz, and 65428 Rüsselsheim, Germany; 2Faculty of Chemistry, A.I. Cuza University of Iasi, Romania; Ion.Laura26@yahoo.com
Native MS & non-covalent Interactions PII38: Characterization of a Cisplatin-DNA-Antibody and its Corresponding Antibody-Antigen-Complexes by high-mass QToF MS and MS/MS Lena Ruhe1, Yves Hachenberger1, Ulrike Hochkirch1, Johanna Hofmann2, Jürgen Thomale3, Michael W. Linscheid1 1 Humboldt-Universität zu Berlin, Deutschland; 2Freie Universität Berlin, Deutschland; 3Universitätsklinikum Essen - Institut für Zellbiologie, Deutschland; lena.ruhe@chemie.hu-berlin.de
PII39: Mass-to-charge-dependent suppression of trapped ions Mario Kompauer, Bernhard Spengler Justus Liebig University Giessen; mario.kompauer@anorg.chemie.uni-giessen.de
Petrolomics, Energy & Combustion PII40: Analysis of crude oil mixtures by Atmospheric pressure photoionization Fourier Transform Mass Spectrometry (APPI-FTMS) Matthias Witt, Arnd Ingendoh Bruker Daltonik GmbH, Bremen, Deutschland; arnd.ingendoh@bruker.com
Cross-Linking PII42: Evaluation of native cross-links in elastin Christoph U. Schräder, Andrea Heinz, Christian E.H. Schmelzer MLU Halle-Wittenberg, Deutschland; christoph.schraeder@pharmazie.uni-halle.de
PII45: Protein structure prediction guided by crosslinking constraints – a systematic evaluation of the impact of the crosslinking spacer length Tommy Hofmann1, Axel Fischer2, Jens Meiler2, Stefan Kalkhof1 1 Helmholtz-Zentrum für Umweltforschung, Deutschland; 2Center for Structural Biology, Vanderbilt University, Nashville; tommy.hofmannn@ufz.de
Proteomics PII41: Characterisation of SIL universal antibody and SIL human proteins for quantitative mass spectrometry Jens Boertz1, Pegah R. Jalili2, James J. Walters2, Gordon Nicol2, Kevin Ray2 1 Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2Sigma-Aldrich, St. Louis, USA; jens.boertz@sial.com
PII43: Influence of glycoforms on the tryptic digestion efficiency of immunoglobulin G based biopharmaceuticals David Falck1, Rosina Plomp1, Bas J. Jansen1, Dietmar Reusch2, Markus Haberger2, Manfred Wuhrer1,3 1 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; 2Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany; 3Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands; d.falck@lumc.nl
PII44: Seeking marker peptides for tracking age-related changes in elastic tissues Angela Cristina Mora-Huertas, Christian E.H. Schmelzer, Christoph U. Schräder, Reinhard H.H. Neubert, Andrea Heinz Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale); angela.mora-huertas@pharmazie.uni-halle.de
PII46: A data independent strategy for a multi-omic approach to investigate obesity treatment within a mouse model Gertjan Kramer1, Nicholas Dekker1, Marc Kipping2, Lee A Gethings3, Victoria Lee4, Robert J Beynon4, James Langridge3, Johannes P.C. Vissers3, Johannes M.F.G. Aerts1 1 Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands; 2Waters GmbH, Deutschland; 3Waters, Manchester, United Kingdom; 4University of Liverpool, Liverpool, United Kingdom; marc_kipping@waters.com
PII47: MS optimization for identification and quantification of TMT labeled peptides Florent Jouy, Stefan Kalkhof, Sina Riemschneider Helmholtz Zentrum fuer Umweltforschung UFZ, Deutschland; florent.jouy@ufz.de
PII48: Complex Regional Pain Syndrome: Targeted Resolvin and Neuropeptide Analysis Anna Schildt1, Tanja Schlereth2, Frank Birklein2, Simone König1 1 Core Unit Proteomics, Interdisciplinary Center for Clinical Research, University of Münster; 2Department of Neurology, University Medical Center Mainz; anna.schildt@uni-muenster.de
PII49: CoFGE mapping of flour proteins for protein aeroallergen database Marina Hanneken, Björn Teichert, Simone König IZKF Core Unit Protemics, Deutschland; marina.hanneken@uni-muenster.de
PII50: Metal labelling for quantification of post translational sugar modifications of proteins Stefanie Ickert, Sebastian Beck, Michael W. Linscheid Humboldt-Universität zu Berlin, Deutschland; stefanie.ickert@chemie.hu-berlin.de
PII51: Improved glycopeptide analysis using acetonitrile enriched sheath gas and oxonium ion dependent ETD Kristina Marx, Andrea Kiehne, Markus Meyer, Zoltan Centnar Bruker Daltonik GmbH, Deutschland; Zoltan.Centnar@bruker.com
PII52: Comparison of peptide separation techniques using strong cation exchange materials Vahid Golghalyani, Marion Bäumlisberger, Ute Bahr, Michael Karas Goethe-Universität-Frankfurt, Deutschland; golghalyani@pharmchem.uni-frankfurt.de
PII53: Investigation of the composition of protein mixtures extracted from muscle tissue with a picoseconds infrared laser Refat Nimer1, Marcel Kwiatkowski1, Sebastian Kruber2, R. J. Dwayne Miller2, Hartmut Schlüter1 1 Department of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Max Planck Institute for the Structure and Dynamics of Matter, University of Hamburg, Germany; r.nimer@uke.de
PII54: Proteome analysis of beewolf-associated symbiotic 'Streptomyces philanthi' revealed bacterial factors essential for survival under in vivo stress conditions. Taras Nechitaylo1, Tobias Engl1, Yvonne Hupfer2, Martin Kaltenpoth1, Aleš Svatoš2, Natalie Wielsch2 1 MPI für Chemische Ökologie, Insect Symbiosis Research Group, Jena, Deutschland; 2MPI Spectrometry/Proteomics Research Group, Deutschland; nwielsch@ice.mpg.de
für
Chemische
Ökologie,
Mass
PII55: Application of Metal Coded Affinity Tagging to Bdellovibrio bacteriovorus René Becker, Sebastian Beck, Michael W. Linscheid Humboldt-Universität zu Berlin, Deutschland; rene.becker@chemie.hu-berlin.de
PII56: Comparison of Thermolysin and Neutrophil Elastase for Peptide Mapping (LC-MS/MS) to fill the Gaps in the Tryptic Digest Björn Mautz, Hans Rainer Völger, Hans Koll, Michael Mølhøj Pharma Research and Early Development (pRED), Large Molecule Research, Dept. of Mass Spectrometry, Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Germany; bjoern.mautz@roche.com
PII57: An assessment of label-free quantification approaches for protein quantification in complex samples Marcus Wurlitzer, Hartmut Schlüter Universitätsklinikum Hamburg-Eppendorf, Deutschland; m.wurlitzer@uke.de
PII58: Investigation of amino acid mutations in the plasma protein C1-Esterase-Inhibitor in a human population Pascal Steffen Universitätsklinikum Hamburg-Eppendorf, Deutschland; p.steffen@uke.de
PII64: Influence of Different Buffer Cations on Charge-State Distribution and Collisional Cross Sections of Native-Like Protein Ions in Native Mass Spectrometry Christoph Hage1,2, Annika Butterer1, Debbie Dewaele1, Frank Sobott1,3 1 Biomolecular & Analytical Mass Spectrometry, Department of Chemistry, University of Antwerp, Belgium; 2Institute of Pharmacy, Department of Pharmaceutical Chemistry & Bioanalytics, Martin-Luther University Halle-Wittenberg; Germany; 3UA-VITO Center for Proteomics, University of Antwerp, Belgium; christoph_hage@web.de
Umwelt - & Lebensmittelanalytik PII59: Discovery of Pesticide Protomers Using Routine Ion Mobility Screening Michael McCullagh1, Davor Turkovic2, Kieran Neeson1, Jeff Goshawk1, David Eatough1, Chris Carver1 1 Waters Corporation, UK; 2Waters GmbH, Deutschland; davor_turkovic@waters.com
PII60: Using the Routine Separation Dimension and Identification Criteria of UPLC Ion Mobility to Enhance Specificity in Profiling Complex Samples. Michael McCullagh1, Davor Turkovic2, Christoph Thomas2, Kieran Neeson1, Jeff Goshawk1, Christopher Carver1, David Douce1 1 Waters Corp., UK; 2Waters GmbH, Deutschland; christoph_thomas@waters.com
PII61: Enhanced reduction of matrix effects using LC-MS/MS with online extraction for the rapid quantitation of antibiotics in milk Louis Maljers1, Arnd Ingendoh2 1 Bruker Daltonics Inc., USA; 2Bruker Daltonik GmbH, Bremen, Deutschland; Arnd.ingendoh@bruker.com
PII62: Verfolgung der photochemisch induzierten Bildung gebundener Rückstände von Benzotriazol mit gelöstem organischem Material mittels ultra-hochauflösender Massenspektrometrie Julia Raeke, Bettina Seiwert, Caroline Davis, Cindy Weidauer, Thorsten Reemtsma Helmholtz-Zentrum für Umweltforschung GmbH - UFZ, Deutschland; julia.raeke@ufz.de
PII63: Eliminating Matrix Effects During Multi-residue Pesticide Analysis by Extensive Dilution Using A New Triple Quadrupole MS With Enhanced Sensitivity Ralf Falter, Andreas Reimann, Thomas Glauner Agilent technologie, Deutschland; ralf_falter@agilent.com
Datum: Mittwoch, 04.03.2015 8:30 9:15 9:15 10:35 10:35 11:00 11:00 12:00 12:00 12:45 12:45 13:00
Plenarvortrag V: Analysis of Protein Binding to Anticancer Metallodrugs Ort: K33
OVIa: Life Sciences & Forensik
OVIb: Hyphenated Techniques
OVIc: Ionisationsmethoden II
Ort: K33
Ort: K32
Ort: I26
Kaffeepause Ort: Foyer
OVIIa: Proteomics III Ort: K33
Abschlussvortrag: Plasma in Hollywood Ort: K33
Tagungsabschluss Ort: K33
OVIIb: Umwelt- & Lebensmittelanalytik OVIIc: Kohlenhydrate II Ort: I26 Ort: K32
Präsentationen Plenarvortrag V Zeit: Mittwoch, 04.03.2015: 8:30 - 9:15 · Ort: K33
Analysis of Protein Binding to Anticancer Metallodrugs Peter O'Connor, Chris Wootton, Peter Sadler University of Warwick, Vereinigtes Königreich; p.oconnor@warwick.ac.uk
OVIa: Life Sciences & Forensik Zeit: Mittwoch, 04.03.2015: 9:15 - 10:35 · Ort: K33
OVIa1: Altersbestimmung von Kugelschreibereinträgen durch Lösungsmittel, Harze und Binder. Andrea Hahn1, Peter Seiler1, Fritz Köhler1, Dieter Kirsch2 1 BKA, Kriminaltechnisches Institut, KT 41 Physikalisch-chemische Urkundenprüfung; 2BKA, Kriminaltechnisches Institut, KT 12 Zentrale Analytik II; Andrea.Hahn@bka.bund.de
OVIa2: Structural investigation of co-assembled clathrin adaptor protein complexes Johannes Heidemann1, Maria Marta Garcia-Alai2, Anna Gieras2, Rob Meijers2, Charlotte Uetrecht1 1 Heinrich-Pette-Institut, Deutschland; 2EMBL Hamburg Unit, European Molecular Biology Laboratory (EMBL), Hamburg, Deutschland; Johannes.Heidemann@hpi.uni-hamburg.de
OVIa3: At the edge of lipidOMICS and proteOMICS: mobility mass spectrometry of lipid-protein adducts Maria Fedorova1,2, Ivana Milic1,2, Zhixu Ni1,2, Eva Griesser1,2, Ralf Hoffmann1,2 1 Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig; 2Center for Biotechnology and Biomedicine, Universität Leipzig; maria.fedorova@bbz.uni-leipzig.de
OVIa4: LUX Score: Cheminformatics Approach to Compute Lipidome Homology Chakravarthy Marella1, Andrew Torda2, Dominik Schwudke1,2,3 1 Research Center Borstel, Deutschland; 2Centre for Bioinformatics, University of Hamburg; 3Airway Research Center North, German Center for Lung Research; cmarella@fz-borstel.de
OVIb: Hyphenated Techniques Zeit: Mittwoch, 04.03.2015: 9:15 - 10:35 · Ort: K32
OVIb1: Investigation of UV transformation products of xenobiotics in the aquatic environment by means of liquid chromatography and mass spectrometry Jörg Roscher, Uwe Karst Westfälische Wilhelms-Universität Münster, Deutschland; joerg.roscher@uni-muenster.de
OVIb2: Electrochemistry coupled to LC/MS for the identification and characterization of reactive xenobiotic metabolites and their protein adducts Tina Wigger1,2, Lars Büter1,2, Uwe Karst1 1 University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 48149 Münster, Germany; 2NRW Graduate School of Chemistry, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany; tina.wigger@uni-muenster.de
OVIb3: Differential Protein Labeling with Electrochemically Generated Reactive Intermediates Lars Büter1,2, Helene Faber1, Tina Wigger1,2, Uwe Karst1,2 1 Institut für Anorganische und Analytische Chemie, Universität Müsnter; lars.bueter@uni-muenster.de
2
NRW Graduate School of Chemistry, Universität Münster;
OVIb4: Study on highly aromatic complex mixtures such as crude oil asphaltenes with online and offline SEC-MS Lilla Molnárné Guricza, Wolfgang Schrader Max-Planck-Institut für Kohlenforschung; guricza@kofo.mpg.de
OVIc: Ionisationsmethoden II Zeit: Mittwoch, 04.03.2015: 9:15 - 10:35 · Ort: I26
OVIc1: Characterization of a versatile low temperature plasma torch by optical emission spectroscopy and time-of-flight mass spectrometry Andreas Bierstedt, Jens Riedel BAM, Deutschland; andreas.bierstedt@bam.de
OVIb2: Mass spectrometry of oligopeptides in the presence of alkali halides using desorption/ionization induced by neutral cluster impact Andre Portz1, Markus Baur2, Julian Heep1, Christoph Gebhardt3 1 Justus-Liebig-Universität Giessen, Deutschland; 2Fakultät Angewandte Naturwissenschaften, Hochschule Esslingen, Deutschland; Daltonik GmbH, Bremen, Deutschland; andre.portz@ap.physik.uni-giessen.de
3
Bruker
OVIb3: Fundamentals and Applications of Plasma-based Ambient Desorption Ionization Mass Spectrometry in Bioanalysis and Lithium-Ion Battery Research Carsten Engelhard1, Anastasia Albert2, Christopher Kuhlmann1, Jacob T. Shelley3, Britta Vortmann2,4, Sascha Nowak2,4 1 University of Siegen, Germany; 2University of Muenster, Germany; 3Kent State University, Kent, USA; 4MEET Battery Research Center, Muenster, Germany; engelhard@chemie.uni-siegen.de
OVIb4: LILBID-MS reveals structural insights into a unique hybrid FoVo rotor and its integration into the ATPase Nina Morgner Uni Frankfurt, Deutschland; morgner@chemie.uni-frankfurt.de
OVIIa: Proteomics III Zeit: Mittwoch, 04.03.2015: 11:00 - 12:00 · Ort: K33
OVIIa1: Identification and relative label-free quantification of lactosylated peptides in bovine raw milk and processed milk products Sanja Milkovska1,2, Ralf Hoffmann1,2 1 Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig; 2Center for Biotechnology and Biomedicine, Universität Leipzig; sanja.milkovska@bbz.uni-leipzig.de
OVIIa2: Multiplexed 2D MRM-based protein biomarker quantitation of plasma proteins from free flaps – a pilot study Jing-zhi Yang1, Andrew Percy2, Jun-Cong Yang2, Christoph Borchers2, Uwe von Fritschen3, Juliane C Finke3, Michael O Glocker1 1 Proteome Center Rostock, University Medicine and Natural Science Faculty, University of Rostock, Rostock, Germany; 2University of Victoria Genome British Columbia Proteomics Center, Vancouver Island Technology Park, Victoria, BC, Canada; 3Division of Plastic Surgery and Hand Surgery, HELIOS Clinic Emil von Behring, Berlin, Germany; jingzhi.yang@uni-rostock.de
OVIIa3: Rapid extraction of intact proteins from tissues via desorption by impulsive excitation Marcel Kwiatkowski1, Marcus Wurlitzer1, Maryam Omidi1, Refat Nimer1, Sebastian Kruber2, R.J. Dwayne Miller2, Hartmut Schlüter1 1 Mass Spectrometric Proteomics, Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf (UKE), Germany; 2Atomically Resolved Dynamics Department ,Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany; m.kwiatkowski@uke.de
OVIIb: Umwelt- & Lebensmittelanalytik II Zeit: Mittwoch, 04.03.2015: 11:00 - 12:00 · Ort: K32
OVIIb1: Flowsystem on-line gekoppelt mit einem LC/qTOF-MS zur systematischen Untersuchung der Photolyse unter umweltrelevanten Bedingungen Bettina Seiwert, Cindy Weidauer, Thorsten Reemtsma Helmholtz-Zentrum für Umweltforschung, Deutschland; bettina.seiwert@ufz.de
OVIIb2: On-line Analysis of Organic Emissions from Residential Wood Combustion with Single-Photon Ionization Timeof-Flight Mass Spectrometry (SPI-TOFMS) Hendryk Czech1, Olli Sippula2,3, Miika Kortelainen2, Jarkko Tissari2, Thorsten Streibel1,3, Ralf Zimmermann1,3 1 Joint Mass Spectrometry Centre University of Rostock, Chair of Analytical Chemistry, Germany and Helmholtz-Zentrum München, CMA (Comprehensive Molecular Analytics), Germany; 2University of Eastern Finland, Department of Environmental Science, Fine Particle and Aerosol Technology; 3Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health (HICE) - www.hice-vi.eu; hendryk.czech@unirostock.de
OVIIb3: Automated fast screening of TOF-MS data Benedikt Weggler1,2,4, Maximilian Jennerwein2,3, Thomas Gröger1,2, Ralf Zimmermann1,2,4 1 aJoint Mass Spectrometry Centre, Cooperation Group “Comprehensive Molecular Analytics” Helmholtz Zentrum Muenchen,; 2GermanybJoint Mass Spectrometry Centre, Institute of Chemistry, Chair of Analytical Chemistry University of Rostock; 3ASG Analytik Service GmbH, Trentiner Ring 30, 86356 Neusäss; 4Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health – Aerosol and Health (HICE); benedikt.weggler@helmholtz-muenchen.de
OVIIc: Kohlenhydrate Zeit: Mittwoch, 04.03.2015: 11:00 - 12:00 · Ort: I26
OVIIc1: Separation of Carbohydrate and Glycoprotein Isomers using Ion Mobility-Mass Spectrometry Johanna Hofmann1,2, Hannes Hinneburg2,3, Heung S. Hahm2,3, Daniel Kolarich2,3, Peter H. Seeberger2,3, Kevin Pagel1,2 1 Fritz Haber Institute of the Max Planck Society, Berlin, Deutschland; 2Institute for Chemistry and Biochemistry, Free University Berlin, Berlin, Germany; 3Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Potsdam, Germany; jhofmann@fhi-berlin.mpg.de
OVIIc2: Influence of glycoforms on the tryptic digestion efficiency of immunoglobulin G based biopharmaceuticals David Falck1, Rosina Plomp1, Bas J. Jansen1, Dietmar Reusch2, Markus Haberger2, Manfred Wuhrer1,3 1 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; 2Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany; 3Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands; d.falck@lumc.nl
OVIIc3: MALDI TOF and ESI IT MS Characterization of highly sulfated Glycosaminoglycan-Oligosaccharides Katharina Lemmnitzer1, Joanna Blaskiewicz2, Jürgen Schiller1, Jörg Rademann2 1 Institut für Medizinische Physik und Biophysik, Medizinische Fakultät, Universität Leipzig; 2Institut für Pharmazie, Freie Universität Berlin; katharina.lemmnitzer@medizin.uni-leipzig.de
Abschlussvortrag Zeit: Mittwoch, 04.03.2015: 12:00 - 12:45 · Ort: K33
Plasma in Hollywood Achim von Keudell Ruhr-Universität Bochum, Deutschland; Achim.vonKeudell@rub.de
Midwinter Reflections on the Progress of Biological Mass Spectrometry Costello, Catherine E. Boston University School of Medicine, USA A long and snowy winter provides a rare opportunity to stop and take stock of where we’ve been and where we’re going. In the twenty years since moving across the river from MIT to Boston University School of Medicine, our laboratory members (and the community at large) have witnessed remarkable changes in our research field, and these have made mass spectrometry much more amenable to addressing the pressing needs of unique bioforms and personalized medicine. Increased sensitivity allows determinations of bioactive peptides in individual patients, topdown sequencing allows assignment of multiple post-translational modifications to individual protein molecules, ion mobility allows structural studies of individual glycoforms and molecular complexes, ultra high resolution allows definition of individual compositions in closely-spaced peaks, MALDI imaging allows monitoring of the spatial distributions of individual components within tissue slices, and new dissociation modes increase the possibility for fully elucidating individual molecular structures. Each of these expanded capabilities has role to play in meeting important, and previously unapproachable, challenges in the detection and treatment of disease. Illustrations from ongoing studies will provide examples of state-of-the-art mass spectrometry in action, and suggest future possibilities.
Ionization at atmospheric pressure: why and how Bruins, Andries University of Groningen, Niederlande Stichworte: ionization, atmospheric pressure, LC/MS, fragmentation, sensitivity The proliferation of atmospheric pressure ionization mass spectrometers was triggered by the invention of electrospray ionization by Fenn and coworkers in 1984. Its development into reliable instruments for everyday use took place at the end of the 1980s and was driven by the need for the coupling of liquid chromatography with MS. All LC/MS systems developed since 1975 were designed for ionization in an electron ionization source or chemical ionization source inside a vacuum envelope. Such designs had to deal with the incompatibility between high liquid flow rates from the LC and the pumping capacity of the vacuum system of the MS. Introduced in 1980, thermospray came a long way to meet the demands of the user, but is was not suitable for continuous use over a long period of time. Horning et al. were the first to build an APCI system for LC/MS in 1974, but although very promising, their design did not lead to a breakthrough. Ionization at atmospheric pressure requires the transfer of ions from atmospheric pressure into the vacuum envelope of the mass spectrometer. Due to strong cooling during expansion into vacuum, a mixture of ions and evaporated solvents will lead to condensation of water and other vapors on sample ions and creation of ion-solvent clusters of unknown size. Examples of designs to avoid this problem include a curtain gas (Sciex) and a heated transfer capillary (Thermo). Inside the vacuum one needs to transfer ions into the mass analyzer, and remove neutrals via vacuum pumps. Separation of ions from neutrals is done by means of RF-only quadrupoles, step-wave lenses, S-lenses and other devices from different manufacturers. The common goal of all such ion guides is to increase the ions-to-gas ratio, and thereby increase sensitivity of the mass spectrometer. Transportation inside the vacuum may be assisted by forward electric fields that accelerate ions. Sensitivity may be increased by higher voltage drops, but the acceleration of ions and collisions with background gas often leads to fragmentation and partial loss of molecular ions. The ion transport discussed so far is independent from the way ions were generated. On the atmospheric side there is a wide choice of ionization methods. The majority of users choose electrospray, either at high flow for general use and high-throughput quantitation, or at low liquid flow rate for proteomics applications. Results obtained by electrospray are very good; most users and several manufacturers ignore the importance of techniques such as APCI and APPI that good alternatives for thermally stable samples. APCI is often sold as an option, rather than as a standard part of an LC/MS system. APCI and APPI are much less susceptible to suppression of sample ionization due to co-eluting matrix components. Examples of ion generation from solid samples are DART, DESI, and a range of related "ambient sampling methods". When we look at the basics of mechanisms of ionization it is clear that electrospray makes use of ions in the liquid phase, while APCI and APPI ionize samples in the gas phase. The "magic" of DART is a combination of rapid heating and evaporation of a sample followed by chemical ionization in the gas phase. Whatever the initial step of ion formation of a sample, what we observe in the mass spectrometer is the final result of interaction of sample ions with background gas composed of air, nitrogen, and solvent vapor in the atmospheric source, and the following passage from atmosphere into the vacuum and into the mass analyzer. Do not be surprised if the spectrum observed does not fit in our simplified ideas of ion stability and reactivity.
How to speed up high-performance mass spectrometry Tsybin, Yury O. (1,2); Nagornov, Konstantin O. (1); Srzentic, Kristina (1); Kozhinov, Anton N. (1) 1: École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; 2: Spectroswiss Sàrl, 1015 Lausanne, Switzerland Ion detection time in FTMS is a substantial cost to pay on the experimental time balance sheet. Extreme complexity of biological samples requires as high throughput as possible, and even then the dynamic range of protein concentration in proteomic samples would not be addressed completely. The situation gets more demanding when ultra-high resolution (>500k in a 200-2000 m/z range) is needed, e.g., as in neutron encoded multiplexed protein quantitation via SILAC approach. Other multiplexed quantitation approaches, e.g., 10-plex tandem mass tag (TMT), may require a moderate resolution of 60k at 100 m/z. However, reaching even this level of performance is time-consuming and throughput-limiting (costs about 100 ms of ion detection on a modern Orbitrap FTMS with absorption mode FT – which is a four-fold increase compared to the widely employed 6-plex TMT approach). New sources of molecular information, e.g., isotopic fine structure of peptides, have shown to be of a value for proteomics, but required resolution performance is not readily achievable on the timescale of a typical proteomics experiment. Similar situation is in the metabolomics in general and in lipidomics in particular applications of high-resolution MS. Finally, biological diversity of diagnostic proteoforms requires mass spectrometry to be performed not on short, 20 µg bis > 100 µg während der Messdauer von 16 s für eine Messung ermittelt. Dann wurden Extraktionsexperimente durchgeführt, bei denen PDMS von Backformen für eine Stunde in Rapsöl bei 180 °C einen Backvorgang simulierte. Dabei wurden Werte von 1 µg PDMS pro mg Öl gefunden. Schließlich wurden reale Backexperimente vorgenommen und Muffins (Mehl, Zucker, Butter, Eier, Backpulver, teils auch Kakao) aus Blechformen mit denen aus Silicon-Backformen verglichen. Von der Oberfläche der Muffins aus Silicon-Backformen war PDMS direkt und deutlich nachweisbar. Die Identität von PDMS wurde anhand der exakten Massen sowie den Isotopenmustern der PDMS abgesichert. Die Ergebnisse deuten stark auf eine Anreicherung von PDMS an bestimmten Stellen der Backwaren. Bei allen Muffins traten zudem meist intensive Signale auf, die [M+NH4]+-Ionen und zugehörigen Cluster-Ionen von Butterfetten zugeordnet werden konnten. Auch die zugehörigen Muffin-Backpapiere wurden überprüft. Sie enthielten große Mengen von Tributylacetylcitrat, einem Weichmacher, der allerdings mit der verwendeten Methode nicht in den Backwaren nachgewiesen wurde. Die Auswahl der geringen Anzahl von Produkten erfolgte zufällig und die hier aufgeführten Werte sollten nicht verwendet werden, um die Produkte exakt dieser Anbieter zu diskreditieren. Vielmehr legen bisherige Ergebnisse nahe, dass alle Artikel aus Silicongummi dieses Verhalten zeigen. Es wird daher angeregt, detaillierte Untersuchungen zur Freisetzung von PDMS aus Silicongummi in lebensmittelanalytischen oder materialprüfenden Laboren anzustellen. Neuer Aspekte Haushaltsartikel aus Silicongummi können beim Gebrauch PDMS-Oligomere freisetzen, die hier mittels DART-FTICR-MS direkt auf der Oberfläche von Backwaren nachgewiesen wurden.
Referenzen [1] Cody RB, Laramee JA, Durst HD (2005) Anal. Chem. 77:2297-2302.; [2] Gross JH (2013) Anal. Bioanal. Chem. 405:8663-8668; [3] Gross JH (2015) J. Am. Soc. Mass Spectrom. DOI: 10.1007/s13361-014-1042-5; [4] Hajslova J, Cajka T, Vaclavik L (2011) Trends Anal. Chem. 30:204-218; [5] Gross JH (2014) Anal. Bioanal. Chem. 406:2853-2862
Studying viral assemblies with mass spectrometry and XFELs Uetrecht, Charlotte Heinrich Pette Institut, Deutschland Stichworte: native mass spectrometry, structural biology, sample delivery, single particle diffraction Einleitung Recent successes in femtosecond X-ray protein nano-crystallography and imaging of single particles demonstrate the prospects of X-ray free-electron lasers (XFELs) for biophysics. Due to the intense and short femtosecond pulses, diffraction patterns of a single particle can be recorded before damaging occurs. Thereby, the major bottleneck in structural biology to obtain large high quality crystals may be overcome. Since the sample is destroyed by the X-ray pulse, a full dataset is acquired in many shots of identical particles. The technique can be used to study structural changes in e.g. protein complexes in a time-resolved manner. Experimenteller Teil However, current sample delivery techniques suffer from an increased background lowering the resolution or are inefficient wasting precious samples. Moreover, the reconstruction of a single structure requires extensive computational sorting. Using native mass spectrometry (MS) for sample delivery, sample consumption would be reduced. Local increased concentrations of a sample can be achieved in ion traps to optimise the interaction rate with the FEL beam. MS is especially attractive for reaction monitoring as minor or transient species can be selected for imaging. Ergebnisse With such an online purification, low populated species can be studied, which would otherwise pass unnoticed. Additional components, like ion mobility or dipole alignment, enable data pre-sorting according to shape and orientation. This could tremendously lower the computational costs to sort diffraction patterns and lead to orders of magnitude shorter calculation times. An introduction to XFELs, sample delivery systems and design considerations for the mass spectrometer are presented. Additionally, most recent native MS data on processing and assembly of non-structural coronavirus proteins will be presented, which form the viral replication complex, and other viral systems my group is currently working on. Neuer Aspekte New method in structural biology
UV-MALDI-MS Analysis of Non-Covalent Streptavidin-Biotin Complexes with a 6-Aza-2-thiothymine Matrix: Effect of Wavelength and Fluence on their Detection Schnapp, Andreas (1); Wiegelmann, Marcel (1); Soltwisch, Jens (1); Dreisewerd, Klaus (1,2) 1: Institute for Hygiene, University of Münster, Germany; 2: Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Germany Stichworte: MALDI-MS, ATT matrix, Non-covalent complexes, Streptavidin-biotin, Laser wavelength Einleitung The analysis of non-covalent complexes by MALDI-MS poses a challenge. Factors that all can result in their unintended dissociation are the acidic character of most MALDI matrices, the requirement of analyte-matrix cocrystallization, and the thermal load imparted during the MALDI process. Best results are frequently obtained with the near pH-neutral 6-aza-2-thiothymine (ATT) matrix [1,2]. Interestingly, this compound exhibits a peak absorption close to 265 nm, far off the standard MALDI wavelengths of 337 and 355 nm. Here, we investigated the influence of the laser wavelength and that of the laser fluence on the ion generation of streptavidin-biotinyl glucagon complexes desorbed from the ATT matrix. Experimenteller Teil Samples were prepared from aqueous solutions using the dried-droplet method. MS experiments were performed with an oTOF-mass spectrometer that was equipped with an optical parametric oscillator laser (OPO; versaScan, GWULasertechnik) and provided tunable laser light with 5 ns pulse duration. Ion signals were recorded for excitation wavelengths between 213-360 nm (step size: 10 nm). The elliptical laser spot size on the target was ~200x400 µm². Ergebnisse Intact non-covalent streptavidin-biotin complexes were detected with optimal ion yields in a wavelength-fluence band between 300 and 340 nm, which is covering the 337 nm emission line of the N2-laser but not that the 355 nm output of the frequency-tripled Nd:YAG laser. With regard to the laser fluence, maximum yields were obtained for fluences exceeding the ion detection threshold by a factor of 3 to 5. In line with our previous results on the detection of gastrin I-peptide-complexes [2], an increased laser penetration depth at these wavelengths of reduced optical absorption on the falling slope of the matrix absorption band appears to support the detection of the complex, while a too low absorption at even higher wavelengths prevents an efficient ionization. The ejection of larger volume elements could lead to an overall process that is similar to IR-MALDI, which is generally regarded as providing “softer” MALDI conditions. Because streptavidin is highly absorbing at lower UV-wavelengths, the second maximum at 240 nm observed for the detection of the non-absorbing gastrin I-peptide complexes is not available. This is presumably caused by direct photodissociation of the streptavidin monomer. For wavelengths between 290 and 300 nm, only the monomers were detected with high signal intensities. Despite of its high affinity (dissociation constant ~10 -15 M [3]) and the effective collisional cooling in the oMALDI ion source, the thermal load acquired within the MALDI process under these conditions appears here as too high to allow for an intact desorption of the complex. The results demonstrate that careful adjustment of all critical sample and irradiation parameters including the excitation wavelength is generally necessary to enable the intact MALDI-MS analysis of weakly non-covalent bound receptor-ligand complexes. Neuer Aspekte Detection of intact streptavidin-biotin complexes by MALDI-MS. Referenzen [1] M. Zehl, G. Allmaier (2003) Rapid Commun. Mass Spectrom. 17: 1931-1940;
[2] A. Schnapp et al. (2014) 47th Annual DGMS Meeting, Frankfurt a. M., 2-5 March 2014, Poster No. PG 3; [3] P. C. Weber et al. (1989) Science 243: 85-88
Fragmentierung von Ethylaminosubstituierten Farbstoffen mittels CID und PD Gernert, Claus; Grotemeyer, Jürgen Christian Albrechts Universität zu Kiel, Deutschland Stichworte: Laserfarbstoffe, Fragmentierung, CID, Fragmentierungsmechanismus Einleitung Coumarine, Oxazine und Rhodamine sind sowohl als Laserfarbstoffe als auch als Label für Photofragmentierungen bekannt. Im Rahmen dieser Arbeit wurde die Fragmentierung unterschiedlicher Farbstofflabel mit Ethylaminoseitengruppen mittels CID und PD untersucht. Für die PD Messungen wurde ein neues Experiment aufgebaut, sodass es möglich ist mittels Nanosekundenpulsen unterschiedlicher Wellenlängen Untersuchungen in der ICR Zelle durchzuführen. Hierbei zeigte sich ein ungewöhnliches Fragmentierungsverhalten der Ethylaminoseitengruppe unter Bildung eines Azarinringes abhängig von der direkten Umgebung der Seitengruppe und der verwendeten Wellenlänge. Experimenteller Teil Alle Messungen wurden mit einem 7.05 T FT-ICR Massenspektrometer der Firma Bruker Daltonik (Bremen) durchgeführt. Die unterschiedlichen Proben wurden mittels Elektrospray Ionisation in die Gasphase überführt, die entsprechenden Ionen in der ICR-Zelle isoliert und anschließend durch CID (Colliosion Induced Dissiociation) mit Argon als Stoßgas sowie PD (Photodissoziation) mit unterschiedlichen Wellenlängen fragmentiert. Hierzu wurde ein Nd:YAG Laser von Continuum zur Fragmentierung mit Nanosekundenpulsen bei einer Wellenlänge von 532 nm, 355 nm sowie 266 nm verwendet. Alle verwendeten Substanzen wurden aus Grundchemikalien von Sigma Aldrich synthetisiert. Ergebnisse Bei den durchgeführten Fragmentierungsreaktionen mittels CID und PD an den substituierten Farbstofflabeln wurden Fragmente sowohl aus der Ethylaminoseitengruppe als auch aus den Farbstofflabeln selbst beobachtet. Neben den erwarteten Fragmenten der Ethylaminogruppe wurden weitere Fragmente beobachtet, die nur unter Beteiligung der benachbarten Alkylgruppen möglich sind. Anhand unterschiedlicher Farbstoffe konnte der Einfluss der Größe sowie der unterschiedlichen Substitutionen untersucht werden, sodass ein zur Bildung der Verluste zugrunde liegender Fragmentierungsmechanismus entwickelt werden konnte. Neben der Abhängigkeit der Abspaltungen von der direkten Umgebung der Seitengruppe konnte auch der Einfluss der Wellenlänge untersucht werden. Insgesamt konnte gezeigt werden, dass die unterschiedlichen Farbstofflabel bei Fragmentierungsuntersuchungen mittels CID und PD viele Abspaltungen bilden und dass Photofragmentierung bei unterschiedlichen Wellenlängen einen großen Einfluss auf die Bildung und Intensität der unterschiedlichen Fragmente hat. Ebenso wie bei den Xanthenfarbstoffen [1] zeigt sich auch bei den substituierten Coumarinen und Oxazinen eine Verletzung der Even Electron Rule, da bei diesen Fällen ebenfalls unterschiedliche Radikale beobachtet werden konnten. Neuer Aspekte Fragmentierung von Farbstofflabeln mittels CID und PD Referenzen [1] J. Peters, J. Grotemeyer, Rapid Commun. Mass Spectrom. 25, 1169-1172 (2011).
Towards a mass spectrometric Western Blot: Multiplexed Targeted Protein Quantification by Gel-LC-MS/MS Gentzel, Marc; Kumar, Mukesh; Drechsel, David; Shevchenko, Andrej MPI of Molecular Cell Biology and Genetics, Dresden, Deutschland Stichworte: Quantitative Proteomics, Targeted Protein Quantification, Gel-LC-MS/MS Einleitung Introduction Quantification of protein abundance changes in biological research is commonly performed by Western Blots or ELISA assays. However, these methods are often impeded by the availability and specificity of antibodies, dynamic range and multiplexing capabilities. Synthesis of isotopic labeled peptides for protein quantification by mass spectrometry is laborious, expensive and inflexible for small-scale discovery studies. We have developed a fast, inexpensive and flexible pipeline for targeted multiplexed protein quantification that relies on the recombinant expression of artificial proteins comprising isotopic labeled reporter peptides for several proteins and Gel-LC-MS/MS. Absolute quantification is achieved by calibration with a quantified protein standard and this method may substitute Western Blots and/or ELISA assays in selected biological and clinical applications. Experimenteller Teil Methods Based on observation of previous label-free LC-MS/MS analyses for each targeted protein the most abundant 4 to 8 tryptic peptides were selected. The peptides were translated into a single artificial gene that was chemically synthesized (Life Technologies). The gene was subcloned into a bacterial expression vector and expressed in a Lys, Arg dual-auxotroph E.coli strain in media complemented with 13C15N-Arg and 13C-Lys (Silantes). The artificial protein was submitted to SDS gel electrophoresis and the protein band was combined with the gel slice containing the protein(s) of interest, co-digested with trypsin in-gel and analyzed by LC-MS/MS on a Orbitrap Velos (Thermo Fisher Scientific) employing the programs Progenesis LC-MS (Nonlinear Dynamics) and/or Xcalibur Qualbrowser (Thermo Fisher Scientific). Ergebnisse Preliminary Results & Discussion Different methods for protein quantification by isotopic labeled peptides (AQUA, [1]), recombinant expression of concatenated peptides [2] or single protein fragments in E. coli [3,4] or wheat germ extracts [5] have been reported previously but have not gained wide-spread application. In our methodical approach each protein is represented by several most abundant tryptic peptides, which improves reliability of quantification. Reporter peptides for multiple proteins are combined into a single artificial gene that is chemically synthesized and available “on-demand”. With high efficiency we have expressed artificial genes comprising more than 30 peptides representing up to 8 proteins and a molecular weight up to 77kD. SDS gel electrophoresis removes the majority of bacterial background and omits the necessity of purification steps and native solubility of the artificial protein. The gel slice containing the proteins of interest and the gel band containing the quantitative standard are combined and co-digested in a single reaction. We have evaluated the capabilities of this method in proof-of-principle experiments. A chimeric protein of 41kD comprising 31 peptides representing 5 proteins was expressed. Full-length expression was confirmed and incorporation of the heavy isotope label was higher than 98%. Five proteins were spiked as an equimolar mixture at different amounts from 20 fmol to 25 pmol per lane into a dominant background of 50µg E. coli lysate. The lysate was separated by SDS-gel electrophoresis, gel slices were excised, combined with gel bands with the chimeric protein, digested with trypsin and respective proteins were then quantified by Gel-LC-MS/MS.
Based on the abundance ratio of “light” and “heavy” peptide counterparts relative quantification of spiked proteins was accurate (R2=0.96) over a dynamic range from 2 fmol to 2.7 pmol of the injected target proteins which enabled their quantification at 0.17-1.94 ng amounts despite dominating (>104-fold) excess of background E.coli proteins. Neuer Aspekte Flexible multiplexed targeted protein quantification by Gel - LC MS/MS. Referenzen [1] Kirkpatrick, D.S. et al. Methods (2005); [2] Beynon, R.J et al. Nat Methods (2005); [3] Brun, V. et al. Mol Cell Proteomics (2007); [4] Zeiler, M. et al. Mol. Cell. Proteomics (2012); [5] Singh, S. et al. J Prot Res (2009)
A universal workflow for efficient enrichment of proteins in mass-spectrometry based proteomics Bonn, Florian; Bartel, Jürgen; Büttner, Knut; Hecker, Michael; Otto, Andreas; Becher, Dörte Institut für Mikrobiologie, Universität Greifswald, Deutschland Stichworte: Protein Enrichment, Proteomics, Solid Phase Extraction, Highly Diluted Samples Einleitung In modern proteomics protein enrichment from diluted samples like body fluids or culture supernatants is a common task. Enrichment is achieved for example by protein precipitation, ultra-filtration or solid phase enrichment (SPE). But all established workflows have certain drawbacks. We have developed an optimized protocol for protein SPE with Strata-Clean beads, leading to bias-free, reproducible results in protein enrichment in very dilute samples. Our protocol [1] is a user-friendly, one-pot method and compatible with GeLC-MS and gelfree workflows. We further have enhanced the scope of StrataClean beads for protein storage on the beads at ambient temperature. Experimenteller Teil We have introduced a priming step for removal of contaminants by acidic hydrolysis. Two workflows were established: a GeLC-MS workflow with optimized protein elution and a gel-free workflow comprising on-bead digest with trypsin. The optimized GeLC-MS workflow was compared with classic techniques. We have demonstrated enrichment from extremely diluted samples (0.1 µg/ml) and unbiased protein purification by LC-MS. Further, we suggest protein storage on the desiccated beads at ambient temperature as a novel application allowing for shipping of samples in a very cost effective way. Ergebnisse Due to quantitative removal of contaminants by bead-priming before protein purification, we assured reproducible and highly efficient protein binding of the StrataClean beads. Several steps of the gel-based workflow were optimized to improve protein elution from the beads. We compared StrataClean enrichment from extremely diluted samples with detergent assisted TCA precipitation and demonstrated highly efficient protein enrichment by a factor of 10,000 with StrataClean in a GeLC-MS experiment. By our new workflow we were able to identify 50 % more proteins reproducible in three technical replicates as with the classic precipitation workflow. With the gel-free workflow we were able to prove bias free protein enrichment after on-bead protein digest. This is a highly interesting option for label-free quantitative proteome studies, including absolute protein quantification. Finally, we established easy protein shipping at ambient temperature. We were able to show that after desiccation of the loaded beads proteins are stable without cooling for more than 10 days. This allows for long range shipping of protein samples at low cost without necessity of elaborated cooling logistics. The combination of highly efficient, bias free protein enrichment, protein purification from extremely diluted samples, GeLC-MS and gel-free analyses and convenient protein shipping at full advantage of the one-pot/ fast protocol of SPE by StrataClean beads makes our new protocol the first universal method for protein enrichment in mass-spectrometry based proteomics. Neuer Aspekte A protocol for superior protein enrichment from highly diluted samples and protein storage at ambient temperature. Referenzen [1] F.Bonn, J. Bartel, K. Büttner, M. Hecker, A. Otto, D. Becher; 2014: Picking Vanished Proteins from the Void: How to Collect and Ship/Share Extremely Dilute Proteins in a Reproducible and Highly Efficient Manner; Anal. Chem. 86; 7421–7427
Characterization of an Improved Ultra-High Resolution Quadrupole Time of Flight (UHR-QTOF) Instrument for Proteomics Applications Kaspar, Stephanie; Lubeck, Markus; Michalski, Annette; Raether, Oliver; Gebhardt, Christoph; Pengelley, Stuart Bruker Daltonik GmbH, Bemen, Deutschland Stichworte: bottom-up, proteomics, QTOF, cell lysate Einleitung In shotgun proteomics it is desirable to identify and quantify a large number of individual peptides from complex samples, such as tryptic digests of human plasma samples or whole cell lysates in the shortest possible time. Complexity and concentration range, however, pose a great challenge to the MS instrumentation in terms of sensitivity, resolution and dynamic range. Several hardware modifications of a bench-top UHR-TOF instrument were carried out and evaluated addressing these particular performance aspects. Experimenteller Teil To test the impact of these modifications on proteomics performance, different complex tryptic digests were analyzed with nano-flow UHPLC and a CaptiveSpray ion source connected to the impact II instrument (Bruker Daltonik). For peptide identification the MaxQuant software package was used (Nature Biotechnology 26, 1367 - 1372 (2008)). Ergebnisse For higher sensitivity at fast acquisition speed, ion extraction from the collision cell into the orthogonal acceleration of the TOF-analyzer was improved by using a novel collision cell design. Increased resolution without changing the effective flight path could be achieved with a modified reflectron. In addition, a faster detector (reduced width of individual ion signals) led to further improvements in resolving power. Comparison with previous design clearly shows an increased resolving power resulting in a full sensitivity resolution (FSR) of 50,000. Using an optimized detector digitizer combination, a threefold higher dynamic range was observed. However in complex samples, the dynamic range is also limited by the capability of the instrument to resolve nearly isobaric compounds. The dynamic range detectable in proteomics samples has been investigated. For one of the most difficult proteomics samples - undepleted plasma - we could cover a dynamic range of 4 orders of magnitude. We will show further results evaluating the impact of the novel hardware features on performance improvements for proteomics applications. Neuer Aspekte Improvements to several hardware components allow identification and quantification of complex proteomics samples with very high dynamic range
Thermal Proteome Profiling Savitski, MIkhail; Reinhard, Friedrich; Drewes, Gerard; Bantscheff, Marcus Cellzome GmbH, Deutschland Stichworte: Quantitative Massenspektrometrie, Targetidentifizierung, Proteomik Einleitung Changes in the thermal stability of proteins are frequently used to study ligand binding and, with the recent development of the cellular thermal shift assay (CETSA), can now be observedin living cells (1), enabling the monitoring of target engagement, which is a key parameter in drug discovery. By combining the CETSA method with multiplexed quantitative mass spectrometry, we established the proteome-wide determination of protein thermal stability in intact cells as an independent and complementary strategy for the characterization of cellular proteotypes. We further demonstrate that monitoring thermal stability across cellular proteomes in different states, such as under drug treatment, enables the identification of direct physical interaction partners and downstream effectors asmarkers of target engagement and drug efficacy. Experimenteller Teil Cells were cultured under differential conditions, such as drug treatment. In an alternative method, the cells were extracted first, and the extracts were treated with drug. For each condition, the cell or cell-extract sample was divided into 10 aliquots. Aliquots were subjected to heating at the indicated temperatures. Samples of intact cells were subsequently subjected to extraction with PBS. After digestion with trypsin, each sample was labeled with a different TMT10 isotope tag. Subsequently, all samples fromeach conditionweremixed and analyzed by means of LCMS/MS.The obtained reporter ion intensitieswere used to fit a melting curve and calculate the melting temperature Tm of each protein separately for the two conditions. Ergebnisse The thermal stability of proteins can be used to assess ligand binding in living cells.We have generalized this concept by determining the thermal profiles of more than 7000 proteins in human cells by means of mass spectrometry. Monitoring the effects of small-molecule ligands on the profiles delineated more than 50 targets for the kinase inhibitor staurosporine.We identified the heme biosynthesis enzyme ferrochelatase as a target of kinase inhibitors and suggest that its inhibition causes the phototoxicity observed with vemurafenib and alectinib. Thermal shifts were also observed for downstream effectors of drug treatment. In live cells, dasatinib induced shifts in BCR-ABL pathway proteins, including CRK/CRKL.Thermal proteome profiling provides an unbiased measure of drug-target engagement and facilitates identification of markers for drug efficacy and toxicity. Neuer Aspekte Neue Massenspektrometrie basierte Methode um die Protein-Bindung bioaktiver Substanzen in Zellen zu messen. Referenzen [1] D. Martinez Molina et al., Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).
Uncovering molecular details of protein "misfolding - aggregation" using affinity- and ion mobility- mass spectrometry: Physiological and Parkinson- Synucleins Przybylski, Michael (1); Lindner, Kathrin (1); Pierson, Nicolas (2); Zhang, Ying (3); Petre, Brindusa-Alina (1,3); Schildknecht, Stefan (1); Gross, Michael (3); Clemmer, David (2) 1: Steinbeis Centzre for Biopolymer Analysis & Biomedical Mass Spectrometry, University of Konstanz, and Rüsselsheim, Germany; 2: Department of Chemistry, Indiana University, Bloomington, USA; 3: Department of Chemistry, Washington University St.Louis, USA Stichworte: Alpha-Synuclein, Physiological and pathophysiological synucleins, oligomerisation-aggregation, ion mobility-MS, affinity-MS Einleitung A large variety of cellular processes are based on the formation and dynamics of multi- and supramolecular protein assemblies, and several diseases, previously thought to be unrelated, such as cancer and neurodegenerative diseases, are characterised by “misfolded” protein aggregates. Chemical structures and reaction pathways of pathophysiological aggregates are only poorly characterised at present. “Soft-ionisation” mass spectrometry (MS), such as HPLCelectrospray-MS, is often unsuitable to direct analysis of reaction pathways and intermediates in aggregation. Recently, ion mobility- MS (IM-MS) has been emerging as a new tool for analysis of protein aggregation due to its concentration-independent gas phase separation capability. Experimenteller Teil Although αSyn and βSyn are closely related with a sequence homology of 62%, βSyn has a different triplet sequence (70-72), VFS, lacks the 11- amino acid stretch Gly73-Glu83 and is devoid of any fragmentation and aggregation. The fragmentation specificity of αSyn motivated us to examine mutants with modified fragmentation and aggregation propensity. Focusing on the substitution of β-sheet- with α-helix- favoring residues, we designed several αSyn mutants with substitutions of three to six aa residues within the hydrophobic aggregation-promoting domain (70-76). All mutant synucleins were prepared by expression in E.Coli and purified by semipreparative HPLC. Ergebnisse First applications of IM-MS to the in vitro oligomerization of α-synuclein (αSyn), a key protein for Parkinson’s disease, enabled the identification of hitherto unknown degradation and aggregation products. Time- dependent studies of the in vitro oligomerization- aggregation of αSyn provided the first identification of a specific autoproteolytic fragmentation, particularly a highly aggregation-prone fragment by cleavage at V71/T72 in the ßbreaking triplett VVT(70-72) in the central aggregation domain [1]. The corresponding recombinant αSyn(72-140) fragment showed substantially faster aggregation and high neurotoxicity compared to the intact protein. The recent development of combined, online bioaffinity-MSmethods [2] enabled first direct (“top-down”) structural studies in vivo, such as from brain homogenate. Applications of affinity-MS will be discussed using epitope-specific αSynantibodies [3] for the characterization of oligomers and interactions in vivo. Most recently, specific mutations of the central (70-72) triplett in synucleins, and affinity-MS provided breakthrough results, such as mutation of the VFS(7072) triplett from physiological ßSyn into αSyn completely abolished neurotoxic aggregation [4].
Neuer Aspekte Ion mobility- and affinity-MS are powerful tools for the molecular elucidation of structures and intermediates of αsynuclein aggregation. Referenzen [1] Vlad, C., Lindner, K., Karreman, C., Schildknecht, S., Leist, M., Tomczyk, N., Langridge, J., Danzer, K., Ciossek, T., Petre, B.A., Gross, M.L., Hengerer, B., Przybylski, M. ChemBiochem. 2011, 12, 2740-2744.;
[2] Slamnoiu, S., Vlad C., Stumbaum M., Moise A., Lindner K., Engel, N., Vilanova M., Diaz, M., Karreman, C., Leist, M., Hengerer, B., Vilaseca, M., Przybylski, M. (2014) J. Am. Soc. Mass Spectrom. 21, 1643-1648.; [3] ] McLaurin, J., et al. (2002) Nature Med. 8: 1263-1269; [4] Przybylski, M. et al. (2014) submitted for publication
Negative Ion Electrospray Tandem Mass Spectrometry of Polyketides Schmidt, Jürgen Leibniz Institute of Plant Biochemistry Halle (Saale), Germany Stichworte: negative ion electrospray tandem mass spectrometry, polyketides, stilbenes, flavonoids, anthraquinones Einleitung Polyketides are widely distributed in plant organisms [1]. During the last twenty years mass spectrometry, especially soft ionization methods as electrospray in combination with tandem mass spectral methods represent an useful tool for structural investigations of such compounds [2-5]. The present lecture deals with the mass spectral behavior of representative model compounds from different important polyketide classes (stilbenes, flavones, isoflavones, flavonols, flavanones, flavanols, phloroglucinols, anthraquinones and bisanthraquinones) under negative ionization conditions. The aim of this study is to compare representative compounds possessing a substituted 1,3-dihydroxy benzene moiety as a common structural feature like resveratrol, apigenin, genistein, kaempferol, naringenin, catechin, emodin and hypericin for a classification of the observed fragmen-tations in a more general way. Experimenteller Teil The negative ion electrospray high-resolution MSn and HCD data of the selected representative model compounds were obtained from an Orbitrap Elite instrument (Thermo Scientific GmbH Bremen, Germany) by direct infusion. Ergebnisse The stilbene resveratrol shows an unusual fragmentation displaying not only an intermediate methyl loss, but also a rearrangement to a naphthol-like fragment ion as indicated by a comparison of the HCD spectra of naphth-1-ol and naphth-2-ol with that of the key ion at m/z 143 obtained by MS2 measurements. In case of resveratrol, apigenin and genistein the loss of carbon suboxide (C3O2) under negative ionization can be assigned as an important fragmentation step indicating the presence of a substituted 1,3-dihydroxy benzene moiety as a common structural feature [2, 4]. On the other hand, in all compounds investigated the prominent unusual loss of CO2 from the [M-H]--ion is of special interest. Other fragmentations being typical for the polyketides discussed are successive losses of ketene (CH 2CO) and CO units as well as specific structure-dependent key fragments. Furthermore, it is demonstrated that high-resolution MS2 and MS3 measurements also allow a differentiation of the origin of isobaric ions. It can be stated that in addition to positive ion electrospray MS/MS data negative ion high-resolution tandem mass spectrometry represents a powerful analytical tool for the structural characterization of such compounds. Neuer Aspekte Comparison of the mass spectral behavior of different polyketide classes under negative ionization to generalize the observed fragmentations Referenzen [1] [1] M.L. Falcone Ferreyra, S.P. Rius and P. Casati: Flavonoids: biosynthesis, biological functions and biotechnological applications. Frontiers Plant Science 3, 1 (2012).; [2] [2] N. Fabre, I. Rustan, E. de Hoffmann, J. Quetin-Leclercq: Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 707 (2001).; [3] [3] F. Cuyckens and M. Claeys: Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 39, 1 (2004).; [4] [4] J.G. Kang, L.A. Hick and W.E. Price: A fragmentation study of isoflavones in negative electrospray ionization by MSn ion trap mass spectrometry and triple quadrupole mass spectrometry. Rapid Commun. Mass Spectrom. 21, 857 (2007).;
[5] [5] E.-H. Liu, L.-W. Qi, J. Cao, P. Li, C.-Y. Li and Y.-B. Peng: Advances of modern chromatographic and electrophoretic methods in separation and analysis of flavonoids. Molecules 13, 2521 (2008).
Combination of proteomic and metabolomic approaches in plant stress research Frolov, Andrej (1,2); Lukasheva, Elena (3); Bilova, Tatiana (1,2); Paudel, Gagan (1,2); Greifenhagen, Uta (1,2); Brauch, Dominic (1,2); Tarakhovskaya, Elena (3); Mittasch, Juliane (4); Osmolovskaja, Natalia (3); Balcke, Gerd Ulrich (5); Milkowski, Carsten (4); Birkemeyer, Claudia (6); Wessjohann, Ludger (7); Hoffmann, Ralf (1,2) 1: Universität Leipzig, Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry; 2: Center for Biotechnology and Biomedicine (BBZ), Leipzig; 3: Saint-Petersburg State University, Faculty of Biology, Department of Plant Biochemistry and Physiology; 4: Martin Luther University Halle-Wittenberg, Interdisciplinary Center for Crop Plant Research (IZN); 5: Institute of Plant Biochemistry, Department of Cell and Metabolic Biology; 6: Universität Leipzig, Faculty of Chemistry and Minerology, Institute of Analytical Chemistry; 7: Institute of Plant Biochemistry, Department of Bioorganic Chemistry Stichworte: AGEs, glycation, post-translational modifications, tandem mass spectrometry, plant environmental stress Einleitung Glycation is a non-enzymatic post-translational modification formed by reaction of reducing sugars (aldoses and ketoses) with amino groups of proteins forming so-called Amadori and Heyns compounds, respectively. Their further oxidation and formation of α-dicarbonyls (glycoxidation) yield advanced glycation end-products (AGEs) known for their pro-inflammatory effects in humans. Though AGEs readily form during thermal processing of foods, it may occur also during the life time of crop plants. In this context, it is important to know the patterns of AGEs in crop plants and effect of environmental stress on their dynamics. Experimenteller Teil The models of high light, drought and metal stress were established with Arabidopsis thaliana, Nicotiana tabacum and Brassica napus. The leaves were harvested before stress application and in multiple points throughout the stress period. The analytical strategy (applied to A. thaliana and B. napus) relied on the combination of LC-based bottom-up proteomics (LC x LC-ESI-Orbitrap-LIT-MS/MS data dependent acquisition experiments), untargeted and targeted metabolomics (GC-EI-Q-MS) and model glycation experiments with synthetic peptides (LC-QqTOF-MS and MS/MS). Ergebnisse Even unstressed A. thaliana and B. napus plants displayed rich patterns of glycated and glycoxidated proteins (up to 386 and 900 modified peptides, respectively), representing mostly regulatory pathways, protein and nucleic acid metabolism. The product structures were comprehensively characterized with ESI-MS/MS. Light and metal stress resulted in increased Amadori/Heyns product formation (mostly triose- and pentose-derived). The number of AGEmodified sites (dominating with arginine residues) was essentially increased only under drought stress conditions (44 unique sequences), while all stresses resulted in significant increase in intensities of corresponding signals. However, the majority of AGE-modified sites did not resemble glycated ones. In contrast, all stresses caused significant increase of tissue carbohydrate contents without elevation of free α-dicarbonyl levels, indicating autoxidation of sugars with immediate binding of α-dicarbonyls to proteins as the most probable scenario. Reactivity towards peptides, glycation and AGE-formation potentials of individual sugars were characterized by in vitro experiments with synthetic peptides. Thereby, D-glucoso-6-phosphate showed the highest reactivity, while dihydroxyacetone phosphate, D-glyceraldehyde and D-ribose demonstrated the highest potential for AGE formation. Neuer Aspekte The stress-related patterns of plant glycation and glycoxidation are characterized for the first time. The mechanisms and precursors proposed.
Online EC/(LC)/ESI-MS for investigating the oxidative metabolism of roxarsone Frensemeier, Lisa; Büter, Lars; Karst, Uwe University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 48149 Münster, Deutschland Stichworte: Electrochemistry, Metabolism, Roxarsone, Arsenic Einleitung Electrochemistry (EC) coupled to electrospray ionization mass spectrometry (ESI-MS) has been proven to be a valuable tool for the simulation of the oxidative drug metabolism. Application of EC/ESI-MS enables the generation and identification of potential phase I metabolites of xenobiotics as well as the investigation of the phase II metabolism and the reactivity of the oxidation products towards biomolecules. In the present study, the metabolism of the arsenic containing drug roxarsone (ROX) is simulated. ROX is an animal feed additive that is frequently used since 1944 and is presumed to be excreted unmetabolized from the animal organism. However, the detailed metabolic pathway is not entirely known and for this reason, toxic effects to humans cannot be excluded. Experimenteller Teil The oxidative metabolism of ROX has been simulated by hyphenation of an EC cell with ESI‑MS. Application of a potential ramp from 0-2500 mV and the presentation of the data in form of a three-dimensional mass voltammogram (MV) enabled the identification of the oxidation products and provided the potential of the highest conversion rate. Addition of a peptide solution (e.g., glutathione (GSH)) directly after the EC cell facilitated the simulation of the phase II metabolism of ROX. By extending the setup with a high performance liquid chromatography (HPLC) system between EC and MS, the reactivity of the oxidation products towards proteins has been investigated. The HPLC system was necessary to remove salts and excessive metabolites from the protein fraction. Ergebnisse The oxidative transformation of the arsenic containing drug ROX has been investigated to simulate the phase I metabolism by means of EC coupled online to ESI-MS. The generation of a mass voltammogram, where mass spectra are plotted against the applied potential, enabled a broad overview on the generated oxidation products. Oxidizing ROX, many different products were detected. Next to mono-, di- and tri-hydoxylated ROX, also aminated compounds as well as the corresponding dehydrogenated species were found. In addition to hydroxylation and amination, a carbon-arsenic-bond cleavage took place and thus, the toxic inorganic species arsenate (As(V)) was formed. Besides the studies of the phase I metabolism of ROX, investigations on the reactivity of the products towards biomolecules were carried out as well. In first experiments, the phase II metabolism was simulated by adding a solution of the tripeptide GSH to the effluent of the EC cell via a T‑piece. In this case, two different adducts with transformation products of ROX could be detected. When the experimental setup was extended by an HPLC system, protein adduct formation was found. For adduct formation experiments, two model proteins were selected: The relatively small, homogeneous whey protein b-LGA and the more complex protein HSA. In both cases, adduct formation with the same oxidation products that showed reactivity towards GSH was observed. Hence, using EC coupled to (LC)/ESI-MS, not only the identification of potential phase I metabolites is possible, but also the estimation of the phase II metabolism as well as the toxicity of the compounds. Neuer Aspekte The oxidative metabolism of ROX was simulated successfully resulting in toxic arsenic species and intermediates that show reactivity towards biomolecules.
Metabolic pathway driven targeted metabolomics – a “quickstep” from mass spectrometric raw data to biologically relevant conclusions Kiehne, Anrdea; Barsch, Aiko; Tellström, Verena; Neuweger, Heiko Bruker Daltonik GmbH, Bremen, Deutschland Stichworte: QTOF, metabolomics, coffee, metabolic pathway Einleitung Biologists often think in terms of biological pathways and try to interpret results derived from Transcriptomics, Proteomics or Metabolomics experiments by making use of these. In addition to the interpretation of known target compounds, scientists in Metabolomics research often also intend to observe significant changes in abundances of as yet unidentified metabolites. Modern full scan high resolution QTOF instruments provide the advantage that both researcher’s requests can be answered using the same data set. In this study a profiling for compounds contributing to the differentiation of coffee according to the assigned strength is performed.
Experimenteller Teil LC-MS data was acquired on a compact QTOF (Bruker) and evaluated using a non-targeted workflow. This nontargeted approach enabled differentiation of coffee types based on their assigned flavor intensity and identified trigonelline (N-methyl-nicotinic acid) as one characteristic compound for weak coffee. We postulated that other compounds contained in metabolic pathways involved in generating trigonelline or derived from trigonelline might have higher abundance in weak coffee as well. Using a novel software tool we queried the elemental composition for trigonelline in the KEGG database (http://www.kegg.jp/). This returned several metabolic pathways involved in trigonelline metabolism. Ergebnisse The nicotinate metabolic pathway was selected for automatically creating a targeted profiling list containing all metabolites of this pathway map. The initial screening list was extended by several compounds known to be characteristic for coffee. The software tool also enabled to quickly screen for the presence of the compounds by generating high resolution Extracted Ion Chromatograms (hrEIC) with narrow mass tolerance window for the target compounds. Interactive views allowed for a quick evaluation of the compounds detected within the samples. The tentative identity of the compounds screened for was substantiated by taking into account accurate mass and isotopic pattern information. For further statistical evaluation all data was exported for PCA calculation. This revealed a clustering according to the coffee intensity as expected based on the previous non-targeted profiling results. The targeted profiling revealed chlorogenic acid and quinic acid as further compounds responsible for differentiating weak and strong coffee, respectively. This statistical targeted evaluation guided the purchase of reference standards for the final confirmation of the identity of chlorogenic and quinic acid. In summary a novel workflow for combined non targeted and pathway driven targeted metabolomics base on the same data high resolution QTOF data files will be presented. Neuer Aspekte Deriving biomarker panels by using knowledge about metabolic pathways after the initial identification of a single marker.
Improving resolving power in complex mixtures by combining tunable synchrotron radiation with advanced mass spectrometric techniques Lucassen, Arnas Physikalisch-Technische Bundesanstalt, Deutschland Stichworte: MBMS, TOF, Isomer Selectivity, Reactiv Mixtures, Multiplexing Einleitung Reactiv flow systems, for instance laminar premixed flames, jet stirred reactors, photolysis reactors, pyrrolisis and oxidation flow reactors or counter flow flames pose a very strong challenge to the mass spectrometric analysis. The Mixture sampled by sample, drawn from this systems by for instance a molecular beam to preserve both the composition at the sampling position and reactive components as for instance radicals, does contain a large set of very similar components. If Fragmentation is not avoided it is virtually impossible to distinguish between contributions from Fragmentation and components with the same mass actually present in the mixture. Experimenteller Teil About a decade ago this was overcome by combining molecular beam time of flight mass spectrometers with tunable Vacuum ultra violet synchrotron radiation. With this technique it is not only possible to avoid fragmentation by ionizing only slightly above the ionization threshold but also to identify isomers by their specific ionization characteristics. Identification and quantification relies on reference ionization data (cross sections and thresholds). This works very well for small molecules even at the low mass resolution caused by the continuous ionization source. For larger molecules, hoever, this fails because of the high number of possible isomers which are than also overlaped by unresolved elemental compositions with the same nominal mass. Ergebnisse With a state of the art orthogonal reflectron time of flight mass spectrometers introduced recently at Sandia National Laboratories, it is now possible to separate species based on their elemental compositions at continuous ionization, which was previously only possible in pulsed techniques. This allows to identify and quantify a much larger set of species unambiguously. In this presentation some specific examples from a variety of systems are shown were new and interesting intermediate molecules in reactions could be identified because of this capability. In larger systems the point where it is impossible to separate different isomers is still reached very quickly. This is especially true for radical species which often have very similar ionization energies and cross sections. Furthermore the basis of reference data is very limited. Many of this limitations can be overcome by Photo electron photo ion coincidence spectrometry. Some results are shown were this technique was recently employed for flame analysis for the first time. Neuer Aspekte Novel combination of high mass resolution with isomer selectivitty in one instrument.
Hochempfindliches Fourier-Transform-Massenspektrometer mit nicht-destruktiver Ionendetektion Schmidt, Michael (1); Brockhaus, Albrecht (1); Butzmann, Stefan (1); Benter, Thorsten (2); Laue, Alexander (3); Aliman, Michel (3) 1: Institut für Sensorik und messtechnische Systeme, Bergische Universität Wuppertal; 2: Institut für Physikalische und Theoretische Chemie, Bergische Universität Wuppertal; 3: Carl Zeiss SMT GmbH Stichworte: breitbandige Ionenfalle, hochauflösend, Ionenspeicherung Einleitung In der modernen Massenspektrometrie werden Quadrupole hauptsächlich für Transferstrecken und als Massenfilter eingesetzt. Quadrupol-Ionenfallen erlauben zudem eine Akkumulation und Beobachtung der Ionen, was diese Instrumente für viele Anwendungen attraktiv macht. Gleichwohl konnten sich Massenanalysatoren, die auf Quadrupol-Ionenfallen basieren, in der Praxis bisher nicht durchsetzen. Die Gründe hierfür liegen in dem begrenzten Auflösungsvermögen sowie niedrigen Messgeschwindigkeiten, verglichen mit FFT-basierten Instrumenten. Diese Nachteile lassen sich jedoch umgehen, indem man die Ionenbewegung – ohne die Ionen zu extrahieren – durch Messung von Influenzströmen detektiert und das Massenspektrum mittels Fourier-Transformation berechnet [1, 2]. Im Folgenden wird ein FFT-basierter Quadrupol-Ionenfallen-Massenanalysator vorgestellt und dessen Leistungsfähigkeit diskutiert. Experimenteller Teil Kernstück des vorgestellten Spektrometers ist eine typische, kompakte Paul’sche Teilchenfalle. Über eine Bohrung in der Ringelektrode lassen sich beschleunigte Elektronen injizieren, mit denen ein Analytgas direkt in der QuadrupolIonenfalle ionisiert werden kann. Ein hochfrequentes Speicherfeld sorgt für eine stabile Speicherung der erzeugten Ionen innerhalb der Zelle. Durch eine geeignete Anregung, beispielsweise einem Rechteckpuls, werden die Ionen aus der Mitte der Ionenfalle heraus beschleunigt. Die dadurch angeregte Ionenschwingungen erzeugen auf den beiden Deckelelektroden Influenzladungen, die über empfindliche und rauscharme Verstärker breitbandig detektiert werden. Mit Hilfe der Fourier-Transformation lässt sich aus dem aufgenommenen Influenzladungssignal das Massenspektrum berechnen. Dabei ist die Influenzladungsmessung grundsätzlich nicht-destruktiv, so dass die Ionen auch nach einer Messung noch stabil gespeichert sind und für weitere Analysen zur Verfügung stehen. Ergebnisse Zur Charakterisierung des Systems werden verschiedene Analyten, darunter unter anderem flüchtige organische Verbindungen (BTX) sowie Toluol in Stickstoff, in niedrigen Konzentrationen in die Teilchenfalle eingelassen und mittels Elektronenstrahl ionisiert. Es lassen sich Konzentrationen im unteren ppb-Bereich nachweisen. Bei Massen unter 200 amu wurde eine Massenauflösung von m/∆m > 18000 erzielt. Da interessante Gaskomponenten häufig nur in Spuren eines dominanten Hintergrundgases vorkommen, z.B. BTX in Luft, sind die Fragen nach Dynamik und Empfindlichkeit von besonderer Relevanz. Hier beweist die Messmethode ihre besonderen Fähigkeiten, denn durch ein vorher berechnetes Anregungssignal können gezielt bestimmte Störkomponenten über eine resonante Anregung aus der Zelle entfernt werden. Da die Ionenpopulationen auch nach Abklingen ihrer Anregung weiter gespeichert bleiben, kann – falls gewünscht – auch mehrfach anregt werden, um eine insgesamt längere Schwingungsdauer und damit eine noch höhere Massenauflösung zu erreichen. Damit vereint das hier vorgestellte System die generellen Vorteile von Ionenfallen mit den Vorteilen gängiger Fourier-Transform-Spektrometer, wie z.B. Ionenzyklotronresonanz-Massenspektrometer oder der Orbitrap. Neuer Aspekte Hochempfindliches und Influenzstrommessung. Referenzen
präzises
Fourier-Transform-Massenspektrometer
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[1] Aliman, M.; Glasmachers, A.: A novel electric ion resonance cell design with high signal-to-noise ratio and low distortion for Fourier transform mass spectrometry, J. Am. Soc. Mass Spectrom. Chem., Oct. 1999, Vol. 10, Issue 10, pp 1000-1007; [2] Laue, A.; Glasmachers, A.: New Design of a Compact Fourier-Transform Quadrupole Ion Trap for High Sensitivity Applications, 57th ASMS Conference; [3] Schmidt, M.; Brockhaus, A.; Brockmann, K.; Benter, T.; Laue, A.; Aliman, M.: Using a Fourier-Transform Quadrupole Ion Trap Operating with Advanced Ion Excitation Methods for High Performance Mass Analysis of Organic Hydrocarbons, 62th ASMS Conference
Coupling thin layer chromatography with mass spectrometry - a practical approach for matrix-loaded samples Griesinger, Hans (1); Matheis, Katerina (1); Schiller, Jürgen (3); Fuchs, Beate (3); Schulz, Michael (2) 1: Merck KGaA, Department of Bioanalytical Chemistry, Deutschland; 2: Merck KGaA, Instrumental Analytics R&D, Deutschland; 3: Universität Leipzig, Institut für Medizinische Physik und Biophysik, Deutschland Stichworte: thin layer chromatography, mass spectrometry, TLC-MS interface, instrumentation, sample preparation Einleitung A straightforward way to couple thin layer chromatography (TLC) with mass spectrometry (MS) is the elution-based TLC-MS interface [1]. It is a semi-automatic system to extract analytes from the TLC plate and transfer them online into the mass spectrometer. It is suitable for all thin layer materials and every eluent that is LC-MS compatible. The interface can be easily connected to any kind of LC-coupled mass spectrometer. Therefore the fields of application are quite broad. In this lecture we will present applications with matrix-rich samples. Experimenteller Teil All experiments were performed on newly developed HPTLC plates with a reduced separation layer thickness. After chromatographic separation the analytes were extracted with acetonitrile/water (95:5, v/v) + 0.1 % formic acid and transferred online into the MS with a flow rate of 0.2 mL/min. Electrospray ionization (ESI) was used in positive and negative mode. Ergebnisse Thin layer chromatography allows sample preparation and chromatographic separation in one step. This is possible because of the high sample matrix tolerance of TLC. Through the coupling of thin layer chromatography with mass spectrometry (TLC-MS) unequivocal substance identification is possible [2]. We show how the elution-based TLC-MS interface can be used for the development of TLC-MS methods for matrixloaded samples in the areas of pharmaceutical ingredients, cosmetic actives and consumer products. Application data is presented for the analysis of methylisothiazolinone (MIT) in wall paint and skin cream. It is shown that the use of thinner TLC plates leads to lower detection limits, increased sensitivity and improved S/N ratios [3]. The sample matrix can be clearly separated from the target analytes. This results in highly unaffected mass spectra due to a very low level of ion suppression. Neuer Aspekte Direct coupling of thin layer chromatography with mass spectrometry via an elution-based TLC-MS interface using newly developed thinner TLC plates. Referenzen [1] Luftmann, H., A simple device for the extraction of TLC spots: direct coupling with an electrospray mass spectrometer, Anal. Bioanal. Chem., 378, 964-968, 2004; [2] Morlock, G., Schwack, W., Hyphenations in planar chromatography, J. Chromatogr. A., 1217, 6600-6609, 2010; [3] Morlock, G.E., Background mass signals in TLC/HPTLC-ESI-MS and practical advices for use of the TLC-MS interface, J. Liq. Chromatogr. Rel. Techn., 37, 2892-2914, 2014
Proton-Transfer-Reaction Mass Spectrometry Coupled with FastGC: High Selectivity in Near Real-Time Sulzer, Philipp (1); Gutmann, Rene (1); Fischer, Lukas (1,2); Herbig, Jens (1); Jürschik, Simone (1); Jordan, Alfons (1); Hartungen, Eugen (1); Hanel, Gernot (1); Märk, Lukas (1); Lanza, Matteo (1,2); Breiev, Kostiantyn (1,2); Märk, Tilmann D. (1,2) 1: IONICON Analytik GmbH., Eduard-Bodem-Gasse 3, 6020 Innsbruck, AUSTRIA; 2: Institut für Ionenphysik und Angewandte Physik, Leopold-Franzens Universität Innsbruck, Technikerstr. 25, 6020 Innsbruck, AUSTRIA Stichworte: FastGC, PTR-MS, TOF-MS, Trace Gas Analysis, Selectivity Einleitung Gas Chromatography (GC) and Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) are two complementary analytical methods. GC on the one side is the gold standard in terms of selectivity and substance identification. However, one drawback of GC is that it is time consuming, which makes real-time analysis impossible. PTR-MS on the other hand has a minimum response time of about 100 ms and thus can be considered as an online and real-time method, but with a somewhat limited selectivity (e.g. no separation of isomers). Here, we present a novel instrumental setup where we combine the advantages of both methods, i.e. we integrate a (fast)GC column into a PTR-MS instrument. This tremendously increases the selectivity but does not considerably limit the PTR-MS' real-time capabilities. Experimenteller Teil PTR-MS is a well established technology that utilizes H3O+, NO+, O2+ or Kr+, respectively, as reagent ions for chemical ionization of trace compounds [1]. The major benefits of this technology are real-time quantification capabilities, high sensitivity (up to 4700 cps/ppbv [2]) and low limits of detection (ppqv range [2]). For this study we modified a PTR-MS instrument by equipping it with a resistively heated fastGC column and a switching mechanism that allows the user to choose between direct injection and fastGC PTR-MS mode. When the instrument is switched to fastGC mode, a sample loop gets filled with the gas entering the instrument's inlet line. Subsequently, the content of the sample loop is injected into the fastGC column. Ergebnisse We exemplify the fastGC process while Manuka tea and spruce resin are analyzed, respectively. In H 3O+ direct injection mode for both samples a peak in the mass spectra appears at m/z 137.13, which can correspond to a multitude of protonated monoterpenes that are not further distinguishable. After switching to fastGC mode the different compounds contributing to m/z 137.13 are separated according to their retention times in the column. The whole process is completed in less than 1 min, which is when the instrument can be switched back to direct injection mode. Thus, by performing an automated fastGC run every 10 to 60 mins (depending on the expected dynamics within the isomers) the two main advantages of SRI-MS and GC-MS are combined: real-time quantification and extremely high selectivity. The fastGC mode offers an additional advantage for PTR-MS measurements, namely the separation of compounds with sample concentrations so high, that the direct inlet PTR-MS analysis would be derogated. Ethanol is such a compound of high abundance in wine head space samples, which would normally require a dilution of the sample. In FastGC mode, the ethanol elutes the column early and the analysis of other compounds at later times thus remains unaffected [3]. We compare the advantages of fastGC PTR-MS in terms of selectivity to those of switching the reagent ions (e.g. from H3O+ to NO+). The latter has already proven to be a valuable tool for substance identification [4] and isomer separation [5] as changing the ion chemistry in the drift tube can lead to the formation of unambiguous product ions. Here we present unpublished data of methyltryptamine isomers. We acknowledge funding through the PIMMS ITN which is supported by the European Commission’s 7th Framework Programme under Grant Agreement Number 287382. Neuer Aspekte
We integrate a fastGC column into a PTR-MS instrument, which considerably improves selectivity but preserves near real-time capability. Referenzen [1] A.M. Ellis, C.A. Mayhew, Proton Transfer Reaction Mass Spectrometry: Principles and Applications, John Wiley & Sons, Ltd., Chichester, UK, 2014.; [2] P. Sulzer, E. Hartungen, G. Hanel, S. Feil, K. Winkler, P. Mutschlechner, S. Haidacher, R. Schottkowsky, et al., International Journal of Mass Spectrometry 368 (2014) 1-5.; [3] A. Romano, L. Fischer, J. Herbig, H. Campbell-Sills, J. Coulon, P. Lucas, L. Cappellin, and F. Biasioli, International Journal of Mass Spectrometry 369 (2014) 81 - 86.; [4] P. Sulzer, B. Agarwal, S. Jürschik, M. Lanza, A. Jordan, E. Hartungen, G. Hanel, et al., International Journal of Mass Spectrometry 354 (2013), 123-128.; [5] M. Lanza, W.J. Acton, S. Jürschik, P. Sulzer, K. Breiev, A. Jordan, E. Hartungen, G. Hanel, L. Märk, C.A. Mayhew, T.D. Märk, Journal of Mass Spectrometry 48 (2013) 1015-1018.
RECENT ADVANCES IN TIME-OF-FLIGHT MASS SPECTROMETRY: WHAT CAN MULTIPLEXING DO FOR YOU? Horner, Gerhard (2); McGregor, Laura (3); BUKOWSKI, Nick (3); Pollack, Leonhard (1); Koschinski, Stefan (1) 1: Markes International GmbH, Deutschland; 2: five technologies GmbH, Deutschland; 3: Markes International Ltd., UK Stichworte: Time-of-flight mass spectrometry, multiplexing Einleitung Time-of-flight mass spectrometry (TOF MS) has made considerable advances in recent years, with the elimination of mass discrimination (crucial for GC couplings), increased sensitivity and compact instrument designs making the technique more amenable. In this presentation, we will discuss advances in TOF MS technology which will further increase its applicability. Experimenteller Teil In reaction kinetics, the simultaneous quantitation of hydrogen with other organics is desirable and can be accommodated by the mass range of modern TOFs. However, the bulk medium (e.g. inert carrier gas) will be present at concentrations many orders higher than species of interest. The term ‘multiplexing’ is used to describe the combination of multiple data streams into a single, comprehensive stream. Multiplexing can be applied to mass spectrometry in a number of ways but, for the purpose of this presentation, we will focus on the use of alternate ‘highpass, low-pass’ filtering of mass and extending the dynamic range of an instrument by alternating the ionisation period between consecutive scans. Ergebnisse The fast switching (up to 100 Hz) between experiments allows extremely low mass range (e.g. m/z 0.5-3.5) to be recorded alternately with a more typical organic mass range (e.g. m/z 10-800), solely eliminating the otherwise detrimental ions - in this case helium - while retaining high time resolution and maximum sensitivity. Switching the ionisation period between consecutive scans can enhance the dynamic range of an instrument by up to 2 orders of magnitude. Fast multiplexing of TOF parameters during acquisition can greatly improve the performance of a TOF MS. Exemplary applications for extending the mass range and the dynamic range of a given instrument will be presented. Neuer Aspekte Fast multiplexing of TOF parameters extends mass range (low mass range and typical organic mass range) and the dynamic range.
Highly time-resolved two-dimensional mass spectrometric imaging of molecular combustion and pyrolysis product concentrations in a burning cigarette Zimmermann, Ralf (1); Streibel, Thorsten (1); Hertz-Schünemann, Romy (1); McAdam, Kevin (2); Liu, Chuan (2); Ehlert, Sven (1) 1: Uni Rostock, Deutschland; 2: BAT R&D, UK Stichworte: photo ionisation, imaging, combustion processes, TOFMS Einleitung Photoionisation mass spectrometry (PIMS) is an established fast on-line analysis technique for organic compounds highly-dynamic combustion processes. A new sampling and data analysis approach allow the mapping/imaging of trace products of combustion e.g. in the tip of a burning cigarette. Experimenteller Teil Photoionisation mass spectrometry (PIMS) is an established fast on-line analysis technique for highly-dynamic combustion processes. A capillary-microprobe sampling system (µ-probe) with PIMS can be used for direct, localized examination of organic vapours from burning or pyrolyzing objects (µ -probe PIMS). This approach now was further developed to a spatial/temporal resolved, in situ imaging method for combustion/pyrolysis products in e.g. a burning cigarette tip. Repetitive experiments were performed at different sampling positions. Ergebnisse
The time resolved PIMS-sequences were combined to spatially resolved, time-dependent “concentration-images” for different compounds. Quantitative images of e.g. nitrogen monoxide-, benzene- and oxygen-consnctrations in the burning tip of a cigarette were measured during a 2-second lasting puff with100ms time resolution. Different formation and destruction zones in the reaction region were observed and time/space-resolved combustion-kinetic data was obtained.
The classical formation/destruction mechanisms of e.g. NO, oxygen and benzene during a cigarette-puff (fuel-NO formation, re-burn, gas phase re-formation via HCN) were space- and time-resolved observed. The data sequence will also be shown as a video sequence, depicting the changes in the spatial combustion product distribution during the 2 sec. lasting, highly dynamic combustion and pyrolysis processes during a single cigarette puff.
Neuer Aspekte neuen Methode (sampling, Datenauswertung) auf Basis der Photoionisationsmassenspektrometrie zum zeit- und ortsaufgelösten mapping/imaging von organischen Verbindungen in Vverbrennungsprozessen, insb.der Feststoffverbrennung
Laserspektroskopische Untersuchung an heterosubstituierten Halogenbenzolen mittels REMPI- und MATISpektroskopie Krüger, Sascha; Grotemeyer, Jürgen Christian-Albrechts-Universität zu Kiel, Deutschland Stichworte: Ionenspektroskopie, Ionisierungsenergie, Anregungsenergie, Schwingungsstruktur Einleitung Ionenspektroskopie ist eine essentielle Methode zur Untersuchung der vibronischen Struktur von molekularen ionischen Spezies und angeregten Neutralen. Es wurden Halogenbenzole bezüglich ihrer vibronischen Struktur des ersten angeregten Zustands (S1) mittels ResonanceEnhancedMultiPhotonIonization (REMPI) Spektroskopie untersucht, der ionische Grundzustand (D0) mit Hilfe der MassAnalyzedThresholdIonization (MATI) Spektroskopie. Ein besonderes Augenmerk widmet sich der Frage, ob aromatische Spezies ihre Planarität bei elektronischer Anregung oder Verlust eines Elektron beibehalten. Die Aufnahme der MATI-Spektren erfolgt in einem Mehrphotonenprozess uber resonante Zwischenzustände. Es wird für jedes untersuchte Molekül ein komplementärer Satz an MATI-Spektren erhalten. Diese Spektren erlauben Rückschlüsse auf die während der Ionisation aktiven Schwingungsmoden. Besondere Aktivität von Schwingungsmoden, z.B. in Form von Progressionen, geben wichtige Hinweise auf Geometrieverzerrungen. Experimenteller Teil Das Experiment besteht aus einem mit einstufiger Ionenquelle und Reflektron ausgerüstetem Time of Flight (ToF)Massenspektrometer [1] und zwei separat (355 nm, Nd:YAG) gepumpten, durchstimmbaren Farbstofflasern. Die DyeWellenlänge beider Laser wird frequenzverdoppelt. Der Aufbau lässt somit Zwei-Farben-Experimente zu. Ein Überschall Molekularstrahl aus Probenmolekülen und Seed-Gas (Argon) wird durch ein gepulstes Jet-Ventil in die Ionenquelle expandiert. Im REMPI-Modus werden die gebildeten Photoionen direkt in das ToF beschleunigt. Im MATI-Modus hingegen wird im Anschluss an die Laserionisation ein schwaches elektrisches Feld angelegt, das die prompt gebildeten Ionen von den Rydberg-Neutralen diskriminiert. Schließlich wird ein Hochspannungspuls angelegt, der die Rydberg-Neutralen ionisiert. Die nach ihrem m/z-Verhältnis separierten Ionen werden durch einen MCPDetektor erfasst. Das Ionensignal wird von einem digitalen Oszilloskop ausgelesen.
Ergebnisse REMPI und MATI Untersuchungen an einer systematischen Reihe von Halogenbenzolderivaten ergaben ein detailiertes Bild der vibronischen Struktur im ersten elektronisch angeregten Zustand (S1) und kationischen Grundzustand (D0) von 1,2-Dichlor-3-fluorbenzol (1,2,3-DCFB), 1,2-Dichlor-4-fluorbenzol (1,2,4-DCFB), 1,3Dichlor-2-fluorbenzol (1,3,2-DCFB), 1,3-Dichlor-5-fluorbenzol (1,3,5-DCFB) und 1,4-Dichlor-2-fluorbenzol (1,4,2DCFB). Für das 1,2,4-DCFB, 1,3,2-DCFB, und 1,4,2-DCFB konnte gezeigt werden, dass es während der elektronischen Anregung zu einer Abweichung der Planarit ẗ entlang der Koordinate einer out-of-plane Mode kommt. Außerdem konnte gezeigt werden, dass es bei diesen Molekülen während der Ionisation aus dem ersten elektronisch angeregten Zustand heraus zu einer Replanarisierung der aromatischen Struktur kommt. Das REMPI-Spektrum von 1,3-Dichlor-2-fluorbenzol zeigt eine ungewöhnlich hohe Aktivität einer niederfrequenten out-of-plane-mode (17b2 ). MATI-Spektren uber verschiedene Moden zeigen eine Verletzung der ∆v=0 Vorzugsregel. Außerdem lässt sich im MATI-Spektrum uber die 17b-Mode eine kurze, dreigliedrige Progression der Mode 17b mit einem Shift des Frank-Condon-Maximums zu niedrigeren Energien beobachten [2] . Das MATI Spektrum uber die out-of-plane Mode (17b2) im 1,2,4-DCFB zeigt sich eine, bisher in der Literatur unbekannte, sechsgliedrige Progression der 17b-Schwingungsmode. Jedes Mitglied der Progression ist zusätzlich in ein Triplett aufgespalten. Desweiteren lieferten oben genannte Untersuchungen sehr genaue Werte für die elektronische Anregungsenergie (AE) und Ionisierungsenergie (IE). Die durchgeführten quantenchemischen Rechnung zeigten gute Ubereinstimmung zwischen Theorie und Experiment. Ebenfalls war mit Hilfe der quantenchemischen Rechnungen möglich REMPI und MATI Spektren zu simulieren.
Neuer Aspekte Geometrieänderung
bei
Anregung
Referenzen [1] F. Gunzer, J. Grotemeyer, Phys. Chem. Chem. Phys., 2002, 4, 5966-5972; [2] S. Krüger, F. Witte, J. Helfrich, J. Grotemeyer, RSC Adv., 2015, 5, 937-948
und
Ionisation
Matrixeffekte bei der LC/MS-Analyse von Vitamin D-Metaboliten mittels verschiedener Ionisierungstechniken Hagenhoff, Sebastian; Hayen, Heiko Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Deutschland Stichworte: DBDI, LC/MS, SPE, Vitamin D, Metabolite Einleitung Vitamin D und seine Metabolite spielen eine essentielle Rolle in der Calcium-Homöostase und sind daher ein wichtiger Faktor für die Knochengesundheit. Die Serumkonzentrationen von 25‑Hydroxyvitamin D3 und anderer Metabolite werden dabei als Biomarker für den Vitamin D-Status herangezogen. Zur Analyse von Vitamin DMetaboliten in humanem Serum wurde die Hochleistungsflüssigchromatographie in Kopplung mit der hochauflösenden Massenspektrometrie (LC/HRMS) eingesetzt. Aufgrund des weiten Polaritätsbereichs der untersuchten Metabolite wurde die dielektrisch behinderte Entladungsionisation (DBDI) [1], welche auf einem Niedriegtemperaturheliumplasma basiert, verwendet und mit etablierten Verfahren zur Ionisation bei Atmosphärendruck (API) verglichen. Experimenteller Teil Zur Anreicherung der Metabolite und Abtrennung von Matrixbestandteilen wurde eine Festphasenextraktion (SPE) der humanen Serumproben durchgeführt. Hierzu wurden die Materialien Waters Oasis ® HLB und Supelco HybridSPE®-Phospholipid Ultra verwendet und miteinander verglichen. Die Ionisationsund Fragmentierungseigenschaften sowie die auftretenden Matrixeffekte bei Einsatz der DBDI, der chemischen Ionisation bei Atmosphärendruck (APCI) und der Elektrospray-Ionisation (ESI) wurden untersucht und verglichen. Ergebnisse Bei der Analyse der extrahierten Serumproben mittels APCI, DBDI und ESI wurden positive Matrixeffekte beobachtet, die vermutlich durch Phospholipide verursacht werden. Diese Matrixeffekte konnten durch die Optimierung der SPE unter Verwendung einer Zirkon-beschichteten Silica-Phase (Supelco HybridSPE®-Phospholipid Ultra), welche Phospholipide entfernt, drastisch reduziert werden. Die DBDI zeigt im Vergleich zur APCI ähnliche Eigenschaften bezüglich der Ionisation und Fragmentierung sowie der analytischen Leistungsfähigkeit. Beide Ionisationsverfahren weisen für höher hydroxylierte Metabolite teilweise intensive Wasserverluste auf, welche zu multiplen Fragmentionen pro Analyt führen. Neuer Aspekte Anwendung der DBDI zur Analyse von Vitamin D-Metaboliten mittels LC/HRMS und Untersuchung der Matrixeffekte. Referenzen [1] H. Hayen, A. Michels, J. Franzke, Analytical Chemistry 2009, 81, 10239– 10245.
Three-layer set-up for standardized quantification of salicylic acid in dried blood spots by desorption electrospray ionization (DESI) mass spectrometry Siebenhaar, Markus (1,2,3); Küllmer, Kai (3); de Barros Fernandes, Nuno Miguel (3); Hüllen, Volker (3); Hopf, Carsten (1,2) 1: Instrumental Analysis and Bioanalysis, Department of Biotechnology, Mannheim University of Applied Sciences; 2: Institute of Medical Technology, University of Heidelberg and Mannheim University of Applied Sciences, PaulWittsack-Str. 10, 68163 Mannheim, Germany; 3: Roche Diagnostics GmbH, Sandhofer Straße 116, 68305 Mannheim Stichworte: Bioanalytical methods, drug monitoring, desorption electrosprayionization, mass spectrometry, pharmaceuticals Einleitung Desorption electrospray ionization (DESI) mass spectrometry has recently been introduced as an emerging technology for direct therapeutic drug monitoring in dried blood spots (DBS) [1,2]. For absolute quantification of various drugs, however, available DBS methods typically require manual application of small molecule internal standards other than the analyte [3]. Aiming to avoid manual handling steps and with industrial standardization in mind, we developed a three layer setup for robust quantification of salicylic acid directly from DBS. Experimenteller Teil A dioctyl sodium sulfosuccinate (DONS) weave facilitating sample spreading was combined with a cellulose layer containing isotope-labeled salicylic acid as internal standard. The third layer is a filter paper that was eventually analyzed by DESI-MS. Using this setup, we developed a quantification method for salicylic acid (SA) from whole blood that covers the entire therapeutic range (30-300 mg/L). Validation of the method was achieved by comparison with LC-MS/MS quantification and results of a cobas® 6000 analyzer. Ergebnisse Compared to previous quantification approaches by DESI MS [2,4], the three-layer setup provided increased sensitivity with a lower limit of quantification (LLOQ) of 20 mg/L SA in whole blood. As a measure of variance, the relative standard deviation (RSD) was used over six sample spots. Quantification experiments showed good reproducibility with RSD ≤ 14% and a correlation coefficient of 0.99. We also showed the applicability to dried plasma spots with a LLOQ of 8 mg/L SA in human plasma. Evaluation of suitability and robustness was achieved by single blind testing. We obtained recovery rates of 95% up to 113% for four samples with RSD ≤ 14%. The RSD of the calibration points was given with < 10% and a correlation coefficient of 0.988.
We successfully tested a three layer setup in DESI-MS applications for introduction of an internal standard into blood samples. This approach obviates the need for any manual sample handling steps. Rapid quantification of drugs or metabolites such as salicylic acid by DESI-MS analysis of DBS on pre-manufactured three-layer cartridges may be a promising approach for future therapeutic drug monitoring, product control or novel MS-based Point of Care devices. Neuer Aspekte novel method for internal standard introduction in ambient mass spectrometry (3 layer method) Salicylic acid quantification on DBS by DESI-MS Referenzen
[1] Li LP, Feng BS, Yang JW, Chang CL, Bai Y, Liu HW (2013) Applications of ambient mass spectrometry in highthroughput screening. The Analyst 138 (11):3097-3103. doi:10.1039/c3an00119a; [2] Wiseman JM, Evans CA, Bowen CL, Kennedy JH (2010) Direct analysis of dried blood spots utilizing desorption electrospray ionization (DESI) mass spectrometry. The Analyst 135 (4):720. doi:10.1039/b922329k; [3] Dias E, Hachey B, McNaughton C, Nian H, Yu C, Straka B, Brown NJ, Caprioli RM (2013) An LC-MS assay for the screening of cardiovascular medications in human samples. J Chromatogr B Analyt Technol Biomed Life Sci 937:44-53. doi:10.1016/j.jchromb.2013.08.01; [4] Kennedy JH, Wiseman JM (2010) Evaluation and performance of desorption electrospray ionization using a triple quadrupole mass spectrometer for quantitation of pharmaceuticals in plasma. Rapid Communications in Mass Spectrometry 24 (3):309–314. doi:10.1002
LMP-EX a multimolecular omics approach for systems biology Ahrends, Robert; Coman, Cristina ISAS, Deutschland Stichworte: lipidomics, proteomics, multi molecular analysis, PPAR signaling Einleitung In order to understand molecular behavior or trends at the systems levels a deep coverage of many molecular species is required. This is especially true for the lipidome, metabolome and proteome, since they are deeply intertwined at different functional layers including signaling, metabolism and cellular integrity. To address this challenge, we introduce here LMP-EX a strategy that allows the quantitative analysis of Lipids, Metabolites and Proteins from one sample with an excellent reproducibility, in an unbiased fashion and with equal efficiency compared to uni-molecular workflows. In summary, the results of this work place LMP-EX as a superior strategy that enables exciting new directions in multiple areas of systems biology ranging from differentiation processes to clinical applications. Experimenteller Teil To achieve this goal we setup a biphasic organic extraction protocol LMP-EX (Lipid, Metabolite, ProteinsExtraction), applied the protocol to lipidomics, metabolomics and proteomics workflows and investigated the applicability of the established protocol to analyses complex systems such as the PPAR signaling system during the onset of adipogenesis. To do so an adaption of a methyl-tert-butyl ether (MTBE)/ methanol lipid extraction protocol [1] was carried out and lipids, metabolites and proteins were subjected to an individual MS based workflow including selected reaction monitoring and high resolution MS and MS/MS experiments. Ergebnisse Here, we established a protocol which can deliver a more comprehensive picture of an entire biological system by extending the detection capabilities to more than one molecular class. With this strategy we were able to access 360 lipids, 70 metabolites and 3327 proteins making this method to the most comprehensive approach for a multimolecular analysis. To illustrate the benefits, of the developed strategy, LMP-EX was applied to the analysis of PPARG signaling in stromal stem cells [2]. Here major molecular remodeling processes for all 3 molecular classes were revealed, which are needed for terminal differentiation into adipocytes. The LMP-EX workflow introduces a novel concept to systems biology allowing for the first time the simultaneous analysis of three different molecular classes from one sample, making this strategy to an excellent and simple approach for systems biology. Therefore this method will supplement common knowledge and identify novel central hubs important for metabolic control. The outcomes of this work have important consequences toward advancing the development of mass spectrometry driven omics workflows as a supportive technique for understanding complex biological systems, in particular for integrative studies combining mass spectrometry data with that from multiple streams, such as genomics, transcriptomics or high content screening approaches. As mass spectrometry instrumentation continuously advances and gains in sensitivity and speed are made, it can be expected that with this established strategy even more molecular species will quantitatively accessible in near future. Neuer Aspekte LMP-EX a multimolecular extraction procedure for quantitative biology. Referenzen [1] Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. Journal of lipid research 49: 1137-1146; [2] Ahrends R, Ota A, Kovary KM, Kudo T, Park BO, Teruel MN (2014) Controlling low rates of cell differentiation through noise and ultrahigh feedback. Science 344: 1384-1389
Analysis of the lipid composition of adipose tissues by spectroscopic methods Popkova, Yulia (1); Meusel, Andrej (1); Breitfeld, Jana (2); Schleinitz, Dorit (2); Hirrlinger, Johannes (3,4); Dannenberger, Dirk (5); Kovacs, Peter (2); Schiller, Jürgen (1) 1: University of Leipzig, Medical Faculty, Institute of Medical Physics and Biophysics, Härtelstrasse 16- 18, 04107 Leipzig, Germany; 2: University of Leipzig, IFB Adiposity Diseases, Liebigstrasse 21, 04103 Leipzig, Germany; 3: University of Leipzig, Medical Faculty, Carl-Ludwig-Institute for Physiology, Liebigstrasse 27, 04103 Leipzig, Germany; 4: Dept. of Neurogenetics, Max-Planck-Institut for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany; 5: Leibniz Institute for Farm Animal Biology, Institute of Muscle Biology and Growth, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Stichworte: Adipose tissue, double bond content of lipids, MALDI MS, NMR spectroscopy, gas chromatography. Einleitung During the last decades many studies focused on the investigation of obesity-related (bio)markers such as adipocytederived peptides [1], but minor interest has been paid to the investigation of the lipids, especially triacylglycerols (TAGs), and fatty acyl compositions of adipose tissues [2]. In this study we combined the strengths of NMR and MS to investigate the impact of the diet on the lipid composition of different adipose fat depots of mice. Beside the localization of the fat (brown, visceral and subcutaneous fat) diet (high fat (HF) vs. standard (SD)) induced differences of lipid composition were of particular interest. All obtained data were also compared with established methods of lipid analysis, particularly thin-layer chromatography (TLC) and gas chromatography (GC) [3]. Experimenteller Teil Animal experiments were performed according to the German guidelines of laboratory animal care. Male mice (n = 6, 3 × standard diet (SD, 11% fat content) and 3 × high fat high sugar diet (HF, 54% fat content); age of 8-12 weeks) were set on diets for 12 weeks. Tissue lipids were extracted using methyl-tert-butyl ether (MTBE) [4]. Adipose tissue lipid compositions were first analyzed by means of MALDI MS (Bruker Autoflex). Qualitative analysis was additionally approved by HPTLC. Quantitative analysis was performed by means of GC (Autosys XL (Perkin Elmer) with a flame ionization detector) and NMR spectroscopy. (1H and 31P HR NMR (Bruker AVANCE-600 at 600.13 MHz for 1H and Bruker DRX-600 at 242.88 MHz for 31P respectively) Ergebnisse Both diets consist almost exclusively of triacylglycerols (TAG), beside minor amounts of phosphatidylcholines (in particular PC 16:0/18:2 (m/z 758.6 and 780.6) and PC 16:0/20:4 (m/z 782.6 and 804.6)) in the "standard" food. However, the overall fatty acyl compositions of the TAGs in both diets differ significantly: the SD is characterized by the presence of TAGs with longer (particularly C18), unsaturated fatty acyl residues (e.g. m/z 901.7 -TAG 54:6), whereas the high fat diet contains nearly exclusively shorter, saturated fatty acyl residues (e.g. m/z 661.5 - TAG 36:0). The lipid composition of the diet supplied to the mice has a strong impact on the lipid composition of the adipose tissues. There are considerable differences depending on the diet and only moderate differences between the types of adipose tissue. All spectra are dominated by the presence of TAGs while there are also some PC species, particularly in brown fat. The observed TAG fatty acyl patterns resemble closely the composition of the SD. In contrast, the adipose tissue extracts at conditions of HF diet are dominated by shorter, saturated fatty acyl residues which are also predominantly present in the HF diet. The relative fatty acyl compositions of both diets and adipose tissues of interest have also been determined by gas chromatography. Longer-chain unsaturated fatty acids (particularly linoleic acid) are nearly exclusively present in the SD, while shorter and saturated ones (particularly lauric and myristic acid) represent the majority of the fatty acids in tissues of mice fed with HF diet. Absolute quantitative data regarding the double bond content were finally obtained by high-resolution NMR spectroscopy, which further supported the above mentioned findings.
In conclusion, the diet composition should be carefully checked prior to performing animal experiments. It is extremely important to use identical diet compositions in order to avoid misinterpretations of experimental results. Neuer Aspekte The incorporation of nutritional fatty acids into tissues was studied by a couple of different spectroscopic methods. Referenzen [1] Saely CH (2012) Brown versus white adipose tissue: a mini-review. Gerontology, 58:15-23.; [2] Hoene M, Li J (2014) The lipid profile of brown adipose tissue is sex-specific in mice. Biochim. Biophys. Acta 1841:1563-1570.; [3] Bielawska K, Dziakowska I, Roszkowska-Jakimiec W (2010) Chromatographic determination of fatty acids in biological material. Toxicol Mech Methods 20:526-537.; [4] Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res 49:1137-1146.
Gender-specific Differences in Blood Plasma Lipidomes of Healthy Females and Males Sales, Susanne (1); Graessler, Juergen (2); Bickle, Marc (1); Cannistraci, Carlo (3); Ekroos, Kim (4); Shevchenko, Andrej (1) 1: Max Planck Institute of Molecular Cell Biology and Genetics, Germany; 2: Department of Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Germany; 3: Biotechnology Center TU Dresden, Germany; 4: Zora Biosciences Oy, Espoo, Finnland Stichworte: human plasma, gender differences, lipidomics Einleitung Lipidomics of human tissues and fluids continuously gains importance for the identification of new biomarkers as risk indicators for a wide variety of diseases. However, due to the differences in analytical methods and biased selection of study populations there are no generally accepted reference values of lipid concentrations. We aimed to define sexspecific reference values and their variance for all major lipid classes and species of human blood plasma. To this end we analyzed a cohort comprising 36 males and 35 females, aged 19 to 33 years, whose clinical blood test and BMI were within clinically accepted ranges. Experimenteller Teil For shotgun lipidomics lipids were extracted by MTBE [1]. Gangliosides were extracted according to Fong et al. [2] with minor modifications. Eicosanoids were recovered by solid-phase extraction as described in [3]. Shotgun analyses were performed on a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a robotic ion source TriVersa (Advion BioSciences) [4]. LC-MSMS analyses were performed on a QTRAP 5500 (AB Sciex). Reference lipid standards spiked in each sample prior extraction were validated by comparison with “quantitative LIPID MAPS standards” supplied by Avanti. Lipids were quantified using LipidXplorer software [5]. Statistical differences between the groups were analyzed using Mann-Whitney U test. Ergebnisse 280 lipid species from 27 lipid classes were quantified by shotgun lipidomics and LC-MSMS analyses. Using non parametric tests we established statistically significant (p Nω-carboxymethylarginine (CMA) > methylglyoxal-derived hydroimidazolon (MGH) > glyoxal-derived hydroimidazolon (Glarg) > Nω-carboxymethylarginine (CEA) > argpyrimidine > pyrraline > dihydroargpyrimidine. Though only few new modification sites appeared with time, essential age-related changes in abundances of glycoxidated proteins were observed: 97 proteins changed their abundance over time (78 of which increased and 19 decreased their abundance). Most of these proteins were involved in signaling and protein metabolism pathways. Surprisingly, glycation patterns were less abundant and comprised arginine-derived Amadori/Heyns products, that was proved by tandem mass spectrometry, to the best of our knowledge, for the first time. As only minimal overlap between early and advanced glycated sites was observed, we propose “oxidative glycosylation” i.e. advanced glycation via monosaccharide autoxidation and carbonyl formation to be the main pathway of AGE formation. Neuer Aspekte Monosaccharide autoxidation was the main AGE formation source in ageing plants. Arginine-derived Amadori/Heyns products were characterized for the first time.
Probing pH-Dependent Protein G´e Surface Topology Alterations by Fast Photochemical Oxidation of Proteins and Mass Spectrometry Yefremova, Yelena (1); Al-Majdoub, Mahmoud (1); Opuni, Kwabena F.M. (1); Koy, Cornelia (1); Yan, Yuetian (2); Gross, Michael (2); Glocker, Michael O. (1) 1: Proteome Center Rostock, University Medicine Rostock, Rostock, Germany; 2: Washington University in St. Louis, St. Louis, Missouri, USA Stichworte: Protein G´e, Fast Photochemical Oxidation of Proteins, ESI-MS, CD-spectroscopy, protein structure changes Einleitung Protein G´e (consisting of three nearly identical IgG-binding domains with linker regions in between and an Nterminal His-tag) is widely used for immobilization and purification of antibodies from body fluids [1]. Antibodies can be released from protein G by dropping the pH from neutral to acidic ranges. In this study we analyzed whether the pH-shift caused structure changes of protein G´e which might be responsible for the antibody release [2]. This was done by using ESI-MS [3] and by Fast Photochemical Oxidation of Proteins (FPOP). In FPOP, hydroxyl radicals rapidly oxidize solvent-exposed amino acid residues. Tryptic digestions and LC-MS/MS analyses of chemically modified peptides pinpoint structural differences with changes in accessibilities of amino acid residues on the peptide level [4]. Experimenteller Teil Protein G´e (10 mM) was dissolved in 50 mM NH4OAc (pH 7) and 2% AcOH (pH 3), respectively [1]. Then 200 mM Gln and 150 mM H2O2 (5 µl, each) were added. FPOP was conducted in a capillary tube at KrF excimer laser power 24.3 mJ/pulse (flow rate: 24.6 μl/min). Reactions were quenched with 10 µl of 200 mM Met and 1 μg catalase. The control sample was handled in the same manner, except it was not laser-irradiated. Oxidation rates were checked on a Bruker MaXis Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Proteins were digested with trypsin/LysC at enzyme to substrate ratios of 1:2, overnight. Peptides were analyzed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher, Waltham, MA) [5]. Ergebnisse A global shift in the charge structure of protein G´e from a native-like (maximum intensity at the [M+10H]10+ ion) to a denatured conformation (maximum intensity at the [M+20H] 20+ ion) was observed by ESI-MS when sprayed from solutions with neutral and acidic pH, respectively. These results agree with CD-spectroscopy of protein G´e. At neutral pH protein G´e shows a predominant α-helical structure, whereas in acidic buffer the CD spectrum of protein G´e indicates lesser secondary structure features. Surface topology alterations at the domain level were mapped by FPOP. Under neutral pH conditions, Met20, Met23, and Met29 (located in the N-terminal flanking region which is not part of the IgG-binding domains of protein G´e) were instantly chemically oxidized. By contrast, oxidations of Trp88, Trp158, Trp228 residues (these are located in the middle of their respective IgG-binding domains and are part of the respective hydrophobic cores) were found in high extent upon FPOP treatment. Similarly, oxidations of Pro113 and Pro183 residues (located in the linker sequences in between the globularly folded IgG-binding domains) were observed as major oxidation events. When switching to acidic conditions, oxidation yields of Met and Pro residues remained unchanged. However, FPOP-induced Trp oxidation decreased as compared to that under neutral conditions. Finally, Tyr48 was found to be oxidized by FPOP exclusively upon acid denaturation. As Tyr48 is not accessible in the native protein structure, oxidation of this residue is explained by unfolding of the protein G´e domain assembly. This opening up of the globular assembly of the three IgG-binding domains with respect to each other could be associated with a simultaneous tightening of the individual domain folds by which hydrophobic areas should be covered and diminish Trp oxidation. In sum, the suggested protein G´e structural changes match with the observed changes in IgG-binding and antibody release under acidic conditions. Neuer Aspekte
Structural changes of protein G´e associated with pH-shift are probed on the peptide level, resulting in a dynamic structure model. Referenzen [1] Yefremova Y, Al-Majdoub M, Opuni KFM, Koy C, Cui W, Yan Y, Gross ML, Glocker MO. “De-novo” amino acid sequence elucidation of protein G´e by combined “top-down” and “bottom-up” mass spectrometry. J. Am. Soc. Mass. Spectrom., in press (2015); [2] Al-Majdoub M, Opuni KFM, Yefremova Y, Koy C, Lorenz P, El-Kased R F, Thiesen HJ, Glocker MO. A novel strategy for rapid preparation and isolation of intact immune complexes from peptide mixtures. J. Mol. Recog. 27, 566-574 (2014); [3] M Przybylski and M O Glocker. Electrospray Mass Spectrometry of Supramolecular Complexes of Biomacromolecules - New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes. Angew. Chem. 108, 878-899 (1996); [4] Hambly DM and Gross ML, Microsecond Time-Scale Hydroxyl Radical Profiling of Solvent-Accessible Protein Residues. In: D. Barcelό, editor: Comprehensive Analytical Chemistry, Vol 52, Protein Mass Spectrometry, J. P. Whitelegge. Elsevier, 151–177 (2009); [5] Yan Y, Chen G, Wei H, Huang RY, Mo J, Rempel DL, Tymiak AA, Gross ML. Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin. J. Am. Soc. Mass. Spectrom. 25: 2084-92 (2014).
Oxidative degradation of Nε-fructosylamine-substituted peptides in heated aqueous systems Greifenhagen, Uta; Sekovski, Viktor; Damjanovic, Marko; Frolov, Andrej; Hoffmann, Ralf Institut für Bioanalytische Chemie, Fakultät für Chemie und Mineralogie; Biotechnologisch-Biomedizinisches Zentrum, Universität Leipzig, Deutschland Stichworte: AGEs, carbohydrates, glycation, synthetic peptides, tandem mass spectrometry Einleitung Protein glycation is a non-enzymatic post-translational modification formed by reaction of reducing sugars with Nterminal and lysyl amino groups. Resulting Amadori compounds readily oxidize ('glycoxidation') [1] yielding advanced glycation end-products (AGEs), a heterogeneous group of compounds demonstrating a clear pro-inflammatory effect in mammals. Alternatively, carbohydrates can oxidatively degrade to α-dicarbonyls, which are highly-reactive towards lysyl and arginyl residues also yielding AGEs ('oxidative glycosylation') [2]. While the latter processes have been studied on protein-modeling peptidesystems[3], AGE formation via Amadori peptide degradation has not been studied. This is, however, important for food quality, as many foods containing Amadori products are heat-treated. Experimenteller Teil The glycated peptides Ac-AKAmadoriASAXFL-NH2 (X = S, H, R, K, D, E) and their unmodified analogues (serving as control) were incubated at 95 °C for up to 4 h. The incubation solution contained phosphate buffer (0.1 mol/L, pH 7.4) without further additives or with FeSO4 (18 or 590 µmol/L) alone or complemented by ascorbic acid (60 µmol/L or 1.2 mmol/L), respectively. RP-HPLC-ESI-QqTOF-MS was used for relative quantification of the analytes by integration of extracted ion chromatograms (m/z ± 0.1). Reaction products were identified by their fragmentation patterns and exact mass (ESI-Orbitrap-MS) and partially confirmed by synthetic standards (identical retention times and fragmentation pattern). Ergebnisse The Amadori compound degraded to less than 10% within 15 min of incubation and was quantitatively lost after 30 min regardless of the incubation condition. The two major products, the unmodified and carboxymethyl (CML)containing peptide, were equally fast formed (70 and 51% of maximum amount at 5 min), suggesting a direct formation from the Amadori peptide, i.e. 'glycoxidation' route to CML. Further evidence was found by (i) the identification of an Nε-(ethanalyl)lysine-containing peptide, an intermediate for CML formation by retroaldol-reaction of the Amadori peptide, which appeared with intermediate kinetics, and (ii) identification of CML only in position 2 (original glycation site) in peptides containing an additional lysyl or argininyl residue in position 6 equally susceptible to the 'oxidative glycosylation' pathway to CML/CMA. Six additional peptides were identified with lysine modifications derived from carbohydrates, and three from oxidation. Oxidation- and carbohydrate-derived products could be distinguished by their formation under different oxidative conditions: whereas carbohydrate-derived products showed increased amounts with higher FeII concentration, oxidation-derived products behaved opposite. Phenylalanine was oxidized to tyrosine independently from the lysine modification. Tyrosine and allysine formation is radical-dependent[4,5] and occurred only to minor or non-detectable amounts in control incubations. Contrary, peptide fragments, observed equally in all incubations, might rather be formed by hydrolysis at labile peptide bonds. Neighboring residues affected both Amadori degradation and product formation. Faster degradation of the Amadori residue by neighboring basic residues did not increase the formation of the two major products. Neuer Aspekte Amadori degradation was studied in a protein-modeling system at 95 °C. Product formation pathways were revealed. Referenzen
[1] M. U. Ahmed, S. R. Thorpe, J. W. Baynes; Identification of N-Epsilon-Carboxymethyllysine as a Degradation Product of Fructoselysine in Glycated Protein. J Biol Chem 1986, 261, 4889; [2] S. P. Wolff, R. T. Dean; Glucose Autoxidation and Protein Modification - the Potential Role of Autoxidative Glycosylation In Diabetes. Biochem J 1987, 245, 243; [3] A. Frolov, R. Schmidt, S. Spiller, et al.; Arginine-Derived Advanced Glycation End Products Generated in Peptide-Glucose Mixtures During Boiling. J Agric Food Chem 2014, 62, 3626; [4] L. M. Dorfman, G. E. Adams, Reactivity of the Hydroxyl Radical in Aqueous Solutions, National Standards Reference Series 1973, 46; [5] E. R. Stadtman ER; Oxidation of Free Amino-acids and Amino-acid-residues In Proteins By Radiolysis and By Metal-catalyzed Reactions. Annu Rev Biochem 1993, 62, 797
Mass Spectrometry Imaging of Biological Tissue: An Approach for Multicenter Studies Römpp, Andreas (1); Both, Jean-Pierre (2); Brunelle, Alain (3); Heeren, Ron M. A. (4); Laprevote, Olivier (5); Prideaux, Brendan (6); Seyer, Alexandre (3); Stoeckli, Markus (7); Smith, Donald F. (8); Spengler, Bernhard (1) 1: Justus-Liebig-Universität Giessen, Deutschland; 2: French Atomic Energy Commission (CEA-LIST), Saclay, France; 3: Centre de Recherche de Gif, CNRS, Gif-sur-Yvette, France; 4: The Maastricht Multimodal Molecular Imaging institute (M4I), Maastricht University, Maastricht, The Netherlands; 5: Chimie Toxicologie Analytique et Cellulaire, Université Paris Descartes, Paris, France; 6: Public Health Research Institute, UMDNJ, Newark, NJ, United States; 7: Novartis Institutes for BioMedical Research, Basel, Switzerland; 8: National High Magnetic Field Laboratory, Florida State University, Tallahassee Florida, United States Stichworte: mass spectrometry imaging, multicenter studies, multimodal imaging, data format imzML, data handling and processing Einleitung Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Experimenteller Teil Here we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the United States. The instrumentation includes MALDI-TOF, MALDI-QTOF, MALDI-FTICR, APMALDI-Orbitrap and Cluster TOF-SIMS. Ergebnisse Experimental parameters such as measurement speed, imaging bin width and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies. Neuer Aspekte Flexible approach to compare and combine MS imaging platforms across different laboratories.
Genaueste Einblicke in die Klimageschichte anhand der Bestimmung von Lipidbiomarkern in marinen Sedimenten mittels LDI FTICR Imaging Wörmer, Lars (1); Alfken, Susanne (1); Elvert, Marcus (1); Fuchser, Jens (2); Lipp, Julius S. (1); Zabel, Matthias (3); Hinrichs, Kai-Uwe (1) 1: Organic Geochemistry Group, MARUM Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, Germany; 2: Bruker Daltonik GmbH, Deutschland; 3: Inorganic Geochemistry Group, MARUM Center for Marine Environmental Sciences and Department of Geosciences, University of Bremen, Germany Stichworte: Imaging, MALDI, Geochemie, Bohrkern, FTMS Einleitung Marine Mikroorganismen passen je nach Habitat und Umgebungstemperatur den Aufbau ihrer Membranen durch Modifikation der Lipidzusammensetzung an. Diese strukturelle Information bleibt auch nach der Sedimentierung erhalten, da die Verbindungen auch über geologische Zeiträume erhalten werden. Daher sind sedimentierte Lipidbiomarker eine wichtige Informationsquelle zur Rekonstruktion der Klimageschichte. Experimenteller Teil Konventionelle Biomarkeranalyse (Extraktion gefolgt von LCMS-Analysen) ist laborintensiv und erfordert Sedimentstücke in cm-Größen, wodurch die zugängliche zeitliche Auflösung häufig niedrig ist. Hier präsentieren wir eine neue Methode, die auf Laser Desorptions Fourier Transformations Massenspektrometrie (FTICR MS) beruht. Diese Methode wurde für die Detektion von Glycerol Dialkyl Glycerol Tetraethern (GDGTs) in Sapropelen aus marinen Sedimenten des östlichen Mittelmeers angewandt. Diese Zellbausteine stammen von einzelligen Meeresorganismen, Archaeen, die sowohl heute als auch damals den weltweiten Ozean bevölkern. In den Sedimenten konserviert, können diese Lipide als sogenannte Proxies genutzt werden. Sie liefern, stellvertretend für direkte Messungen, Informationen über vorzeitliche Meeresbedingungen und werden in den meisten Fällen repräsentativ als Proxy für Meereswassertemperaturen eingesetzt[1]. Ergebnisse Durch das Abrastern eines Sedimentkerns mittels LDI FTICR MS werden die relativen Intensitäten der GDGTs direkt aus Sedimentkernen gewonnen und können mit der zum Zeitpunkt der Sedimentierung herrschenden Meeresoberflächentemperatur korreliert werden[2]. Die hohe räumliche Auflösung des Lasers (hier: 250 µm) in Kombination mit dem extremen MS-Auflösungsvermögen des FTICRs ist die Voraussetzung zur Gewinnung relevanter Daten. Da die resultierenden Massenspektren sehr komplex sind, wird zur Beschränkung des Massenbereichs ein Quadrupol zum Vorfiltern benutzt. Diese neuartige Methode ermöglicht somit die Untersuchung vorzeitlicher Klima- und Umweltveränderungen in bislang unvorstellbarer zeitlicher Auflösung. Neuer Aspekte Hochaulösendes MALDI imaging von geologischen Bohrkernen zur Erhöhung der zeitlichen Auflösung von Daten für die Klimageschichte Referenzen [1] Schouten, S., Hopmans, E.C., Schefuss, E., Damste, J.S.S., 2002. Distributional variations in marine crenarchaeotal membrane lipids: a new tool for reconstructing ancient sea water temperatures? Earth and Planetary Science Letters 204, 265-274.; [2] Wörmer L, Elvert M, Fuchser J, Lipp JS, Buttigieg PL, Zabel M, Hinrichs KU (2014). Ultra-high-resolution paleoenvironmental records via direct laser-based analysis of lipid biomarkers in sediment core samples. Proc Natl Acad Sci USA 111 (44), 15669-156
Combining label-free whole-cell MALDI MS biotyping and imaging mass spectrometry for in-situ monitoring of histone deacetylase drug target activation Munteanu, Bogdan (1,2); Meyer, Björn (1,2); Burgermeister, Elke (3); Reitzenstein, Carolina v. (1,2); Ebert, Matthias P. (3); Hopf, Carsten (1,2) 1: Center for Applied Research in Biomedical Mass Spectrometry (ABIMAS); 2: Instrumentelle Analytik und Bioanalytik Hochschule Mannheim; 3: Universitätsklinikum Medizinische Fakultät Mannheim der Universität Heidelberg Stichworte: Whole-Cell MALDI MS, Target engagement, Histones, Tumor models, Imaging Einleitung Epigenetics describes the heritable changes in gene expression that do not require changes in nucleotide sequence. Reseach revealed that cancer is not only a genetic diseases but can also result from a deregulation in epigenetic enzymes e.g histone deacetylases (HDAC)(1). HDAC inhibitors are focus of research. Drug binding and target activation are prerequisites of efficacy and need to me monitored during drug development. State of the art techniques require reagents and multiple preprocessing steps and reagent-free in-situ analysis of engaged drug targets or targetproximal pharmacodynamic signatures in solid tumors remains challenging. MALDI biotyping (2) demonstrated that HDACi potencies can be label-free quantified in whole-cells and MALDI-IMS revealed the distribution of acetylated histones and thus confirming the tumor-selective pharmacodynamic responses (2). Experimenteller Teil Application of Whole-Cell MALDI MS biotyping to detect histone core proteins under whole cell conditions without performing acid extraction or LC-purification. Monitoring the pharmacodynamic fingerprint of HDAC inhibitor(HDACi) drug target activation and evaluation of HDACi drug potencies on pharmacodynamic histone fingerprints. Developement of a proteins MALDI- mass spectrometry imaging technique to visulize the histone acetylation state in solid tumors and to monitor the proximal biomarkers of HDACi drug target activation in a gastric cancer mouse models. Ergebnisse Histone Core proteins can be directly detected from whole cells by Whole-Cell MALDI MS without prior acid extraction and purification, by increasing the MALDI matrix acidity. Whole-Cell MALDI MS is suitable to study the target engagement of pharmacutical drugs (HDACi) under whole-cell conditions and the label-free evaluation of their IC50 values relying on histone acetylation-sepecific mass shifts. By applying MALDI mass spectromery imaging on the histone mass shifts the spatiotemporal distribution of acetylated histones and thus the tumor-selective pharmacodynamic responses in a mouse model of gasterointestinal cancer could be confirmed. Taken together, the combined power of Whole-Cell MALDI biotyping and MALDI mass spectrometry imaging suggets that the monitoring of drug-induced mass shifts could provide a suitable platform in pharmacology, clinical oncology and drug discrovery. Neuer Aspekte Quantification of HDACi on their pharmacodynamic fingerprint. In-situ analyis of drug target activation of HDACi in solid tumor models. Referenzen [1] Gastroenterology. 2012 Jul;143(1):99-109.e10; [2] Anal Bioanal Chem. 2012 Nov;404(8):2277-86.; [3] Anal Chem. 2014 May 20;86(10):4642-7
High precision mass measurement and separation of nuclear isomers with a multiple-reflection time-of-flight mass spectrometer Hornung, Christine (1); Ayet, Samuel (1,2); Dickel, Timo (1,2); Ebert, Jens (1); Geissel, Hans (1,2); Haettner, Emma (2); Knöbel, Ronja (2); Miskun, Ivan (2); Pietri, Stephane (2); Plaß, Wolfgang R. (1,2); Purushothaman, Sivaji (2); Reiter, Moritz Pascal (1); Rink, Ann-Kathrin (1); Weick, Helmut (2); Dendooven, Peter (3); Diwisch, Marcel (1); Greiner, Florian (1); Heiße, Fabian (2); Jesch, Christian (1); Kalanta-Nayestanaki, Nasser (2); Lang, Johannes (1); Lippert, Wayne (1); Moore, Iain (4); Pikhtelev, Alexandre (5); Pohjalaine, Ilkka (4); Prochazka, Andrej (2); Ranjan, Manisha (3); Scheidenberger, Christoph (1,2); Takechi, Maya (2); Winfeld, John S. (2); Xu, Xiaodong (1,2); Yavor, Mikhail I. (6) 1: JLU Gießen, Germany; 2: GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany; 3: KVI, University of Groningen, 9747 AA Groningen, The Netherlands; 4: University of Jyväskylä, FI-40014 Jyväskylä, Finland; 5: Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Russia; 6: Institute for Analytical Instrumentation, Russian Academy of Sciences, 190103 St. Petersburg, Russia Stichworte: Time-of-flight mass spectrometer, Multiple-reflection, Nuclear isomer separator, Exotic nuclei Einleitung Nuclear isomers are excited metastable states of nuclei. Their long lifetimes originate from their shape, spin or spin projection. The properties of nuclear isomers can differ from the ground state properties besides their half-life e.g. their decay properties. Applications of nuclear isomers affect a broad field of research including new possibilities for clean energy storage to impact on nuclear astrophysics and the production of the elements in the universe. Particularly in the region of exotic nuclei, with an extreme neutron-to-proton ratio, these excited states can only be produced, measured and separated under challenging conditions. The multiple-reflection time-of-flight mass spectrometer (MRTOF-MS) [1,2] is an ideal tool to provide new information and opportunities. Experimenteller Teil The MR-TOF-MS has been developed as part of the FRS Ion Catcher [3] at GSI in Darmstadt. Here exotic nuclei are produced at relativistic energies and are separated in-flight in the fragment separator (FRS). They are slowed down and thermalized in a cryogenic stopping cell, before they are transported in a radio frequency (RF) quadrupole beam line towards the MR-TOF-MS where mass measurements and ion separation are performed. In the MR-TOF-MS [4] the ions are accumulated, cooled and injected in bunches by a linear RF trap. In the analyzer they are reflected multiple times to enlarge their flight path by orders of magnitude to obtain high resolving powers in a very short measurement duration. Ergebnisse The performance of the MR-TOF-MS was improved. The kinetic energy of the ions in the time-of-flight section of the MR-TOF-MS has been increased to 1300eV. Mass resolving powers (FWHM) for 133Cs of 120,000, 220,000 and 420,000 have been obtained in 2.3 ms, 4.6 ms and 18.3 ms, respectively. A novel RF quadrupole-based switchyard was recently developed and commissioned. It provides the opportunity to additionally inject calibration ions into the RF beam line by merging beams from up to 5 beam lines. Projectile and fission fragments of Uranium at the FRS produced at 1000 MeV/u were measured for the first time directly with a MR-TOF-MS at low yields (few ions per hour), among them 213Rn with a half-life of only 19.5 ms. Due to the single ion sensitivity of the device, it can perform these mass measurements. In the region of the chart of nuclei of the doubly magic nucleus 132Sn several nuclei have long-lived nuclear isomers. Ground and excited state of 133I were measured simultaneously with the MR-TOF-MS. This allows the direct determination of directly the mass of both states and therefore the excitation energy of the isomeric state. Furthermore the isomeric ratio can be measured at the same time. This will lead to a better understanding of the production mechanism of exotic nuclei at accelerator facilities.
The MR-TOF-MS is not only suited for mass spectrometry. It is also used, equipped with a Bradbury-Nielsen gate, as an isobar and nuclear isomer separator. In the MR-TOF-MS the ground and excited state of an exotic ion can be separated spatially. This was demonstrated in an experiment in October 2014. With the opportunity to produce a pure ion beam which consists of one excitation state only, new possibilities arise in the novel field of mass resolved decay spectroscopy in nuclear physics. Neuer Aspekte High resolution MR-TOF-MS with single-ion sensitivity enable measurement and separation of nuclear isomeric states of rare short-lived nuclei. Referenzen [1] W.R. Plaß et al., Int. J. Mass Spectrom. 349, 134-144 (2013); [2] T. Dickel et al., Nucl. Instrum. Methods A doi: http://dx.doi.org/10.1016/j.nima.2014.12.094 (2015) (in print); [3] W.R. Plaß et al., Nucl. Instrum. Methods B 317, 457-462 (2013);; [4] W.R. Plaß et al., Nucl. Instrum. Methods B 266, 4560-4564 (2008);
Analysis of polar trace components in middle distillate fuel by ultra-high resolution mass spectrometry using ESI and GC-APCI Rüger, Christopher Paul (1); Smit, Elize (2); Sklorz, Martin (1); De Goede, Stefan (3); Zimmermann, Ralf (1,4); Rohwer, Egmont (2) 1: Joint Mass Spectrometry Centre / Chair of Analytical Chemistry, University of Rostock, Rostock, Germany; 2: Department of Chemistry, University of Pretoria, Pretoriam South Africa; 3: Sasol Technology, Fuels Research, Sasol, Sasolburg, South Africa; 4: Cooperation Group Comprehensive Molecular Analysis, Helmholtz Zentrum München, Neuherberg, Germany Stichworte: Diesel, ESI, GC-APCI, FT-ICR-MS Einleitung Fossil fuel analysis is still a challenging analytical task, even for light and middle distillate fuels. Polar compounds, such as oxygenated or heterocyclic species, are especially interesting due to their influence on physical properties, e.g. corrosivity or viscosity [1,2]. State-of-the-art techniques are chromatographic techniques, such as gas-chromatography (GC) and comprehensive 2-D GC (GCxGC) [3]. Typically these instruments are equipped with time-of-flight mass analyzers, supplying excellent acquisition speed, but usually lacking in resolving power. In the presented work we applied high-resolution mass spectrometry with classical direct infusion electrospray ionization (ESI) in positive and negative mode to cover basic and acidic species. Furthermore a hyphenation of GC to atmospheric pressure chemical ionization (APCI) was used for the determination of medium polar compounds. Experimenteller Teil Four reference (Europe-EN590, California, Swedish-MK1, USA-US2D) and nine commercial diesel samples were investigated. The polar fraction of each sample was extracted by mixing fuel with methanol. For analyzes analyses with ESI the extract was further diluted with methanol. For GC-APCI-analyses the neat fuel diluted in a methanol/toluene-mixture was injected. Mass spectrometric analyses were carried out using a Bruker-solariX-FT-MS and Bruker-apex-FT-MS (both equipped with a 7T magnet) for ESI and GC-APCI respectively. For (+/-)-ESIanalyses 200 scans were accumulated, a resolving power of roughly 150000 @ m/z 200 was obtained and each sample was analyzed in triplicate. For the GC-APCI-analyzes a BPX5-column was used and a resolving power of roughly 340000 @ m/z 200 was obtained with a scan frequency of 0.8Hz. Ergebnisse Using ESI, the reference diesel samples showed a completely different pattern in -class distribution when compared to that from the commercial diesel samples, e.g. in positive polarity mode the CHN1-class was significantly more abundant in the commercial diesel samples. This behaviour can be partially explained by considering the origin of the samples, i.e. blending and distillation. Very low intensities for the standard diesel indicate a low content of polar species, leading to a higher percentage variation between the replicates. In negative polarity mode mainly compounds with oxygen and/or sulphur were found, whereas in positive mode mainly pure hydrocarbons and nitrogen containing species were observed. Nevertheless some compound classes, such as CHN1 and CHOx, were found in both data sets, but a different pattern for the double bond equivalent distribution was obtained. Therefore it can be assumed that different functional groups were detected for species with the same elemental composition. The GC-APCI-hyphenation enables isomeric/isobaric differentiation and minimises matrix effects. Even at high acquisition rates the chromatographic features cannot be distinguished, but some information can be gathered based on the retention time index (RI). Sophisticated data analyses by self-written MATLAB routines compared measured RI values and data from the NIST database for limiting proposed elemental compositions. As a result the +1O 1 subclass showed 13 peaks when summing up all mass spectra for the GC run to mimic direct infusion, whereas 42 species were detected using the retention time resolved data processing. This multidimensional information was visualized by using a variation of the traditional Kendrick plot, where GC retention times are used instead of nominal mass values on the x-axis. By applying this visualization-technique the standard diesel samples can be well separated from the commercial diesel samples.
Neuer Aspekte - investigation of polar compounds in diesel fuels using (+/-)-ESI and (+)-APCI - use of GC-APCI hyphenation to FT-ICR-MS for diesel analysis Referenzen [1] Anastopoulos G, Lois E, Karonis D, Kalligeros S, Zannikos F. Impact of oxygen and nitrogen compounds on the lubrication properties of low sulfur diesel fuels. Energy 2005;30:415–26.; [2] Balster LM, Zabarnick S, Striebich RC, Shafer LM, West ZJ. Analysis of Polar Species in Jet Fuel and Determination of Their Role in Autoxidative Deposit Formation. Energy Fuels 2006;20:2564–71.; [3] Phillips JB, Xu J. Comprehensive multi-dimensional gas chromatography. Journal of Chromatography A 1995;703:327–34.
Adding a chemical dimension to the analysis of heavy crude oils with ultra-high resolution mass spectrometry Wang, Xuxiao; Schrader, Wolfgang Max-Planck-Institut für Kohlenforschung Stichworte: Ultra-high resolution, mass spectrometry, chemical method, electrospray ionization, crude oil. Einleitung Fossil-based materials including heavy crude oil are still the main supply of energy. The presence of even only a small percentage of non-hydrocarbon elements (such as N-, S-, and O-) in crude oil is deleterious and harmful for refining catalysts and environments. Crude oil is considered the most complex mixture in nature. Chemical methods in combination with ultrahigh resolution mass spectrometry (the high-field Orbitrap FTMS and FT-ICR MS) coupled with ESI provide an effective and feasible approach to selectively analyze the nonpolar sulfur species in whole crude oils without any fractionation and chromatographic separation. Experimenteller Teil DBT was dissolved in dry 1, 2-dichloroethane (DCE) and was reacted with a certain amount of deuterated derivatization reagents (C2H5I) in the presence of silver tetrafluoroborate (AgBF4) for 4 h. The precipitate was removed by centrifugation and washed with dichloromethane. The same procedure was further applied to a mixture of standard aromatic CH-, N-, O- and S-containing compounds and a heavy bitumen. C2H5I was replaced by CD3I and C2D5I in heavy bitumen reaction. Mass spectra were recorded using a 7 T FT-ICR MS (Thermo Fisher Scientific, Bremen) and a high-field Orbitrap FTMS equipped with an ESI source. External mass calibration was performed using a tune mix solution. Ergebnisse As known, electrospray ionization (ESI) coupled with ultrahigh resolution mass spectrometry is an excellent method to selectively analyzing polar compounds; nevertheless, most of components of crude oil are non- or less-polar. Herein, we introduce the chemical methods to crude oil to selectively analyze the S-containing species under positive ESI conditions. DBT was selected as an example to optimize the reaction condition while the reaction was monitored by 1H NMR analysis. These optimized conditions were then employed to a mixture of standard aromatic CH-, N-, O- and Scontaining compounds, DBT, anthracene (ANTH), dibenzofuran (DBF) and acridine (ACR), and after reaction, only DBT was selectively tagged. The same conditions were finally introduced into the real heavy bitumen, and normal C2H5I was replaced by the deuterated alkylation reagents (CD3I and C2D5I), by which, the specific information of D atoms which are not present at high abundance in crude oil allows unambiguously discriminating the nonpolar sulfur species by CD3 or CD5 group from original crude oil in positive ESI. As a reference, the original heavy bitumen was prepared with the solvent DCE containing 0.2 % AcOH, and polar N- and NS-species were observed by protonation. The results show that the S1- and S2-species were selectively tagged showing a DBE range (double bond equivalent) from 2- 25. Coordination chemistry, nucleophilic substitution together with relevant methodology play role in selective analysis of sulfur-containing compounds. Chemical methods in combination with the ultrahigh resolution mass spectrometry coupled with ESI provide an effective approach to selectively analyze the nonpolar sulfur species in whole crude oils without any fractionation and chromatographic separation. Neuer Aspekte Combining chemical reactions with ultra-high mass spectrometry is used to selectively analyzing crude oil.
Improving cross-linked peptide identification in large protein complexes across different high-resolution LC/MS/MS platforms Lenz, Christof (1,2); Lee, Chung-Tien (1,2); Dybkov, Olexandr (1); Lührmann, Reinhard (1); Urlaub, Henning (1,2) 1: Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Deutschland; 2: Universitätsmedizin Göttingen (UMG), Deutschland Stichworte: cross-linking, protein complex, high-resolution MS Einleitung Most cellular proteins do not function in isolation, but assemble into higher order complexes to perform their physiological roles. Conventional high-resolution structural methods such as NMR or X-ray crystallography show intrinsic limitations in the analysis of large or dynamic protein complexes. To overcome these, chemical cross-linking combined with mass spectrometry (XL-MS) is applied more and more to identify spatially proximal protein components, thereby generating distance constraints that can be used to generate or refine structural models. We have optimized workflows to apply XL-MS to Megadalton protein complexes, and will discuss improvements and limitations regarding (i) sample preparation and separation, (ii) mass spectrometric analysis on different highresolution MS platforms, (iii) data processing and (iv) implementation into structural modeling. Experimenteller Teil Protein complexes of different sizes were chemically cross-linked with bissulfosuccinimidyl suberate (BS3), reduced, alkylated and tryptically digested into (cross-linked) peptides. Additional separations were performed either on the protein (SDS-PAGE) or peptide (SEC, RP-HPLC) level to enrich cross-linked species. The final samples were analysed by nanoLC/MS/MS using ion-trap and beam-type CID on several instrument platforms, including different Orbitrap (Thermo Q Exactive, Q Exactive HF, Orbitrap Fusion) and Quadrupole-Time of Flight mass spectrometers (AB SCIEX TripleTOF 5600+). Data analysis was performed using pLink 1.18 and 2.0 beta software (Chinese Academy of Sciences) [1], and searching against custom-made protein sequence databases. Cross-links were tested for plausibility by matching against existing structural models where possible. Ergebnisse The success of XL-MS studies of large protein complexes depends on careful optimization of experimental parameters. Whereas cross-linked complexes consisting of few components can be separated from non-cross-linked proteins by SDS-PAGE, larger complexes have to be trypsinized in solution. Here, peptide/peptide cross-links can be enriched from the large excess of non-cross-linked or “dead-end” peptides e.g. by size exclusion chromatography (SEC). Optimization of nanoLC/MS/MS conditions revealed that both general instrument properties and parameter optimization contribute significantly to the confident identification of larger numbers of cross-links. MS/MS acquisition speeds and MS/MS spectral quality are of major importance, as even after enrichment peptide/peptide cross-links are a minority in the complex mixtures analysed. With regard to CID activation, beam-type, highresolution CID (HCD) significantly outperformed ion-trap CID. In addition, optimization of the CID collision energy provided significant improvements irrespective of the instrument generation used. Another hurdle towards the routine use of XL-MS experiments for structural biology lies in the database search algorithms used for the identification of cross-linked species. While significant improvements have been implemented, there is still a lack of reliable statistical means for results evaluation such as an unbiased, global False Discovery Rate (FDR) analysis, resulting in a significant need for manual evaluation of large datasets. We tested the plausibility of database search results by matching identified cross-links including their scores, numbers of spectral counts and the resulting distance constraints against available structural models generated by highresolution X-ray crystallography for smaller, well-characterized protein complexes. We found that a surprisingly high percentage of the identified crosslinks matched within plausible tolerance, demonstrating the validity of the approach. Application of the improved overall methodology on Megadalton-sized protein/protein complexes resulted in the
identification of >1000 crosslinks for individual complexes, highlighting the need for statistical tools to validate the results in a more automated fashion. Neuer Aspekte Chemical cross-linking and mass spectrometric analysis (XL-MS) for the structural analysis of large protein complexes Referenzen [1] Yang B et al., Nat. Methods 9(9), 904‐6 (2012).
Monitoring Conformational Changes in PPARβ/δ by Chemical Cross-Linking, Photo-Affinity Labeling, and Mass Spectrometry Schwarz, Rico; Tänzler, Dirk; Kölbel, Knut; Ihling, Christian; Sinz, Andrea MLU Halle-Wittenberg, Deutschland Stichworte: Bpa, photo-affinity labeling, BS²G-D0/D4 Einleitung Chemical cross-linking, combined with an enzymatic digestion and mass spectrometric analysis of the reaction products, has evolved into an alternative strategy to identify protein-protein and protein-ligand interactions [1]. Peroxisome proliferator-activated receptors (PPARs) belong to the subfamily of nuclear receptors that are involved in metabolic processes. One subtype, PPARβ/δ, is thought to be connected with the development of several chronic diseases and presents an important drug target [2]. Experimenteller Teil The ligand-binding domain (LBD) of PPARβ/δ (amino acids 166-441) was cross-linked with the amine-reactive homobifunctional cross-linker bis(sulfosuccinimidyl)glutarate (BS²G)-D0/D4. For photo-affinity labeling (PAL) studies, several variants of the LBD of PPARβ/δ, in which specific amino acids were exchanged against p-benzoyl-Lphenylalanine (Bpa) [3], were cross-linked using a home-built UV-irradiation chamber. After in-gel or in-solution digestion, cross-linked samples were analyzed by nanoHPLC/nano-ESI-MS/MS with an UltiMate 3000 RSLC system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex ion source (Thermo Fisher Scientific). Cross-links were evaluated with the software StavroX [4]. The distance constraints imposed by the cross-links served to monitor conformational changes in PPARβ/δ upon binding of the agonists GW1516 and GW0742. Ergebnisse Cross-linking experiments with BS²G indicate a high flexibility of the LBD of PPARβ/δ. Both in the absence and in the presence of ligands, the majority of cross-links involved amino acids 185-210. In the presence of the agonists GW1516 and GW0742, additional cross-links were found from the N-terminus of the LBD to lysines K322 and K422/K423 suggesting a large conformational change. To confirm this, additional cross-linking experiments with fulllength PPARβ/δ are currently performed. To obtain additional information about conformational changes induced by ligand binding, PAL studies are in progress. For these experiments, the phenylalanine residues F177, F189, F210, and F245 as well as the C-terminal tyrosine Y441 were exchanged against Bpa. All site-specific mutations were validated by MS/MS experiments. One cross-link was found in all experiments connecting Bpa177 to isoleucine I378, as both residues are located on opposite helices. Surprisingly, Bpa cross-links were often identified as two distinct species even before MS/MS experiments were conducted: One species is the intact cross-linked product, one species shows the loss of one water molecule. This finding indicates that after Bpa cross-linking the hydroxyl group is eliminated already in solution resulting in the formation of an alkene. CID, HCD, and ETD fragmentation experiments of Bpa cross-linking products are currently performed. Neuer Aspekte Chemical cross-linking, photo-affinity labeling, conformational changes in PPARβ/δ upon ligand binding Referenzen [1] Sinz, A.; Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and proteinprotein interactions, Mass Spectrom Rev (2006); [2] Berger et al.; The Mechanisms of Action of PPARs, Annu Rev Med (2002); [3] Schultz et al.: A chemical toolkit for proteins-an expanded genetic code, Nat Rev Mol Cell Biol (2006);
[4] Götze et al.; StavroX-A Software for Analyzing Crosslinked Products in Protein Interaction Studies, J Am Soc Mass Spectrom (2012)
Chemical crosslinking of TSHR-ECD Nagel, Marcus M. B. (1,2); Schaarschmidt, Jörg (1); Meiler, Jens (3); Kalkhof, Stefan (2); Paschke, Ralf (1) 1: University of Leipzig, Division of Endocrinology and Nephrology; 2: Helmholtz Centre for Environmental Research – UFZ, Department of Proteomics, Leipzig, Germany; 3: Vanderbilt University, Center for Structural Biology, Nashville, USA Stichworte: Chemical Crosslinking, Orbitrap-MS, TSHR, computational modelling, protein structure Einleitung Chemical crosslinking is a powerful tool for the elucidation of protein structures especially those that cannot be solved by classical approaches like X-ray or NMR. The major advantages of this method are its high sensitivity and the possibility to study low purity proteins. Furthermore, experiments can be executed under native-like conditions [1]. The Thyroid Stimulating Hormone-Receptor (TSHR) is a Glycoprotein Hormone-Receptors (GPHR), which is of high interest for the fundamental understanding of membrane proteins and potential medical applications. A particular characteristic of GPHRs is the large Extracellular Domain (ECD) that includes the glycohormone binding site which is of special interest [2]. The aim of this study is to create the first experimental based model of the TSHR-ECD. Experimenteller Teil Cross-linking reactions were performed using DST (disuccinimidyl tartrate), BS 3 (bis(sulfosuccinimidyl) suberate), sulfo-EGS (ethylene glycol bis(sulfosuccinimidylsuccinate)), BS(PEG)5 (bis(succinimidyl) penta(ethylene glycol)). All cross-linking reactions were conducted in 1X PBS-buffer, pH 7.2 at a protein concentration of 3.3 or 2.5 µM. The protein to cross-linker ratio was 1:100 or 1:200 (n:n) respectively and the incubation was carried out at room temperature for 60 or 120 min before the reaction was quenched with an equimolar amount of ammonium bicarbonate buffer. Afterwards, the proteins were in-gel digested with trypsin. MS-measurements were performed with an LTQOrbitrap XL (Thermo Fisher Scientific Inc.) online coupled to an NanoAcquity‑UPLC (Waters Corporation) or an Orbitrap Fusion Tribrid-MS (Thermo Fisher Scientific Inc.) online coupled to an UltiMate 300 HPLC (Dionex Corporation). Ergebnisse For decades the structure of TSHR and especially the TSHR-ECD have been studied by scientists, but still no atomistic model of this protein is available. Much of the insights into the structure of the GPHR are based on the crystal structure of the Follicle stimulating Hormone-Receptor (FSHR)-ECD [3], which has a moderate sequence identity of ~40% to the TSHR-ECD [4]. This enabled our working group to create homology models of the TSHRECD which could be filtered and probed using our crosslinking data. In total, we identified 41 inter- and intramolecular crosslinks (XL). Six intramolecular crosslinks (XL) were detected in the unbound state of the TSHR-ECD, of which, four represented the C-terminal part, which was not covered by any structural model so far. Furthermore, we found 22 XLs for the TSHR when bound to TSH. Two were intramolecular XLs for the TSHR-ECD and 10 were intermolecular XL between the bound TSH and its receptor. These XLs delivered valuable information about the relative orientation, position and interaction between the two proteins and their respective structures. While 18 XLs of the TSHR-ECD in complex with TSH matched perfectly the best-scored predicted TSHR structure, four XL did not fulfill the criterion stating that distance between the Cβ-atoms should be smaller than the sum of the spacer length plus the side chains of the two connected amino acids. However, three of these four divergent XL were located on the protein terminus, which was predicted to be highly flexible. Interestingly, the remaining XL, obtained with DST fits better in a second modelling cluster (22.4 Å in maximum) and also depends strongly on the chosen template for the modelling process. By applying chemical crosslinking in combined with MS and molecular modeling our working group was able to deliver an experimental based model of the TSHR-ECD.
Neuer Aspekte First experimental based structure model for the GPCR TSHR-ECD are obtained using chemical crosslinking and computational modelling. Referenzen [1] A. Sinz, Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and proteinprotein interactions, Mass spectrometry reviews 25 (2006) 663–682.; [2] T. Davies, R. Marians, R. Latif, The TSH receptor reveals itself, The Journal of clinical investigation 110 (2002) 161–164.; [3] Q.R. Fan, W.A. Hendrickson, Structure of human follicle-stimulating hormone in complex with its receptor, Nature 433 (2005) 269–277.; [4] G. Vassart, L. Pardo, S. Costagliola, A molecular dissection of the glycoprotein hormone receptors, Trends in biochemical sciences 29 (2004) 119–126.
Incorporation of Photo-Methionine into Calmodulin for Photo-Cross-Linking/MS Studies Piotrowski, Christine; Ihling, Christian; Sinz, Andrea Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther University HalleWittenberg Stichworte: Photo-Cross-Linking, Photo-Methionine, E. coli Einleitung Photo-cross-linking is an auspicious tool to obtain 3D-structural information of a protein and to gain insights into protein-protein interactions. The advantage of photo-cross-linking is that different amino acid side chains can be targeted, while the mainly used chemical cross-linkers prefer to react with primary amine or hydroxy groups [1]. If photoactivatable amino acids are incorporated into the proteins of interest, no external cross-linker has to be added [2]. Here, we present the successful incorporation of the photo-reactive diazirine analogon of methionine into the 17-kDa protein calmodulin (CaM), which is a highly conserved Ca2+‑binding signaling protein that plays an important role in a variety of cellular processes. CaM contains nine methionines that can potentially be replaced by photo-methionine (pMet) [3]. Experimenteller Teil For the incorporation of pMet into CaM, E. coli was grown in mineral salts medium supplemented with pMet during overexpression of the recombinant protein. His6-tagged CaM was enriched from cell extracts using immobilized metal ion affinity chromatography and size exclusion chromatography. Cross-linking experiments were performed in 20 mM HEPES buffer (pH 7.4) with a free Ca2+ concentration of 30 nM. The photo-cross-linking reaction was induced by UV-A irradiation (360 nm, 8000 J/cm2). Mass spectrometric analysis of cross-links was done after in-solution digestion with trypsin and GluC by nano-HPLC/nano-ESI-MS/MS on an Ultimate 3000 RSLC nano system coupled to an Orbitrap Fusion Tribrid mass spectrometer. Data were analyzed with the StavroX software 3.4.9 [4]. Ergebnisse The incorporation of pMet into CaM is straight-forward as the translation machinery accepts pMet instead of methionine. Therefore, no additional components, e.g. tRNAs, were required for the expression of pMet-labeled CaM. After purification of pMet-labeled CaM, the successful incorporation of pMet was validated by peptide fragment fingerprint MS analysis. In all samples, all nine methionines of CaM were found to be replaced by pMet. Calculation of the pMet incorporation rate was performed for CaM samples obtained at different timepoints after induction. The highest pMet incorporation rate ( >30%) was obtained at 3h after induction. Obtained pMet-labeled CaM was used for photo-cross-linking experiments resulting in the identification of several intramolecular cross-links within CaM, in which different pMet residues (positions 36, 76, 144 and 145) are involved. The identified cross-links are in agreement with the proposed structure of CaM at low Ca2+ concentration and demonstrate that short valuable distance information can be obtained by the incorporated photoactivatable amino acids and subsequent photo-cross-linking. Neuer Aspekte Incorporation of pMet is easily performed in E. coli using a mineral salts medium without incubation on complex media. Referenzen [1] Sinz, A. (2014) The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks, Expert review of proteomics 11, 733-743.; [2] Suchanek, M., Radzikowska, A., and Thiele, C. (2005) Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells, Nat Methods 2, 261-267.; [3] Vetter, S. W., and Leclerc, E. (2003) Novel aspects of calmodulin target recognition and activation, Eur J Biochem 270, 404-414.;
[4] Götze, M., Pettelkau, J., Schaks, S., Bosse, K., Ihling, C. H., Krauth, F., Fritzsche, R., Kühn, U., and Sinz, A. (2012) StavroX-A Software for Analyzing Crosslinked Products in Protein Interaction Studies, Journal of the American Society for Mass Spectro
Mass spectrometric analysis of post-translational modifications in T cells Freund, Christian Freie Universität Berlin, Deutschland Stichworte: tyrosine phosphorylation, sortase-mediated ligation Einleitung Post-translational modifications of proteins are dramatically alter their function. They often act that reflect the "on" or "off" state of a protein Therefore, trapping the state of these modifications conditions represents an important task in modern proteomics research.
ubiquitous and can as molecular switches within the living cell. under certain cellular
Experimenteller Teil - SILAC -18O/16O labeling [1] - Affinity purification [2] - Quantiative mass specgrometry [3,4] -
Sortase
mediated
ligation
[5]
Ergebnisse Here, we follow two key modifications of biological systems, namely palmitoylation and phosphorylation, by mass spectrometry. For palmitoylation, we use established acyl-biotin exchange chemistry to map the subset of proteins that is reversibly palmitoylated in primary T cells from healthy human donors. Similarily, phopsho-proten interactions can be mapped by the combination of affinity purification, chemical labeling and quantitative mass spectrometry. Therefore, our approach offers a powerful mean to infer protein function from biologically complex samples Neuer Aspekte Phosphorylation-dependent interactome mapping of "structured" phosphosites Sortase-mediated ligation for affinity-MS Palmitome and phosophoproteme analysis of human primary T cells Referenzen [1] Lange, S., Sylvester, M., Schümann, M., Freund, C. & Krause, E. (2010). Identification of phosphorylationdependent interaction partners of the adapter protein ADAP using quantitative mass spectrometry: SILAC vs (18)Olabeling. J. Proteom. Res. 9, 411; [2] Lehmann, R., Meyer, J., Schuemann, M., Krause, E. & Freund, C.* (2009). A novel S3S-TAP-tag for the isolation of T-cell interaction partners of adhesion and degranulation promoting adaptor protein. Proteomics 9, 5288-5295; [3] Kofler, M., Schümann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A. Schaefer, M., Lührmann, R., Krause, E. and Freund, C.* (2009). Proline-rich sequence recognition I: Marking GYF and WW domain assembly sites in early spliceosomal complexes. M;
[4] Schlundt A, Sticht J, Piotukh K, Kosslick D, Jahnke N, Keller S, Schuemann M, Krause E, Freund C.* (2009). Proline-rich sequence recognition II: proteomic analysis of TSG101-UEV interactions. Mol. Cell. Proteom. 8, 24742486; [5] Kuropka B, Royla N, Freund C, Krause E. (2014) Sortase A-mediated site-specific immobilization for identification of protein interactions in affinity purification-mass spectrometry experiments. Proteomics, epub ahead of print.
Lipids originating from Mycobacterium tuberculosis (Mtb) are detected in plasma of Tuberculosis Patients Zehethofer, Nicole (1,2); Heyckendorf, Jan (3); Schaible, Ulrich E. (1); Lange, Christoph (3); Schwudke, Dominik (2) 1: Research Center Borstel, Cellular Microbiology, Parkallee 1-40, Borstel, Germany;; 2: Research Center Borstel, Bioanalytical Chemistry, Parkallee 1-40, Borstel, Germany; 3: Research Center Borstel, Clinical Infectious Diseases, Parkallee 1-40, Borstel, Germany Stichworte: LC-MS, tuberculosis, lipidomics, high-resolution mass spectrometry Einleitung The human plasma lipidome is highly complex. While some “shotgun lipidomics” approaches rely solely on high resolution mass spectrometry for lipid identification1, tandem mass spectrometry is required to specifically identify fatty acid residues bound to particular lipid species2. Odd-numbered fatty acid residues are of low abundance in human plasma. However,Mycobacterium tuberculosis (Mtb)3, the causative agent of tuberculosis (Tb), contains oddnumbered fatty acids in high abundance. To address the question whether a tuberculosis infection can be detected based on the analysis of phospholipids containing odd-numbered fatty acid residues in human plasma samples, we developed a method to specifically identify these species in plasma. This will allow for identification of potential mycobacterial-derived lipid species in plasma samples of infected individuals. Experimenteller Teil Plasma samples were obtained from Tb patients before and during anti-Tb treatment and from healthy controls. 50µl plasma was extracted using methyl-tert-butyl ether and normal phase-HPLC was used for separation of lipid classes. Online detection was performed using a high resolution hybrid Apex-Qe FT-MS system (Bruker Daltonics, Bremen). Mass spectra were summed up according to the respective retention time ranges of the lipid classes of interest, smoothed, baseline subtracted and peak lists were exported using customized VBA scripts (DataAnalysis 4.0). Lipids were assigned using LipidXplorer if the mass error was better than 3ppm4. Tandem mass spectrometry experiments were performed on a Q Exactive Plus (Thermo Fisher Scientific, Bremen) for identification of the fatty acid composition of the major phospholipid classes. Ergebnisse Proof-of-principle experiments were performed using a small subset of patient samples (n=3) since study enrolment is still ongoing because anti-Tb treatment requires six months for completion. By using the described LC-MS approach, we were able to quantify more than 300 lipid species in Tb patients’ plasma samples. Identified lipid species were analysed using targeted tandem mass spectrometry experiments for identification of phospholipid species containing odd-numbered fatty acids. We were able to quantify more than 50 lipid species containing odd-numbered fatty acids in plasma samples obtained from Tb patients and from healthy controls, accounting for ~ 2 mol% of total identified plasma lipids. In this subset of lipid species identified, we were particularly interested in phospholipid species containing the mycobacterial specific tuberculostearic acid (TSA). In mycobacterial lipid extracts, TSA is primarily found in its esterified form, where it is incorporated into phosphatidylinositol species containing saturated fatty acids as the second fatty acid residue, for example PI(19:0/16:0). In plasma lipid extracts of Tb patients, we found that TSA is mainly incorporated into phosphatidylinositol and phosphatidylethanolamine species containing poly unsaturated fatty acids as second fatty acid residue. Neuer Aspekte Detection of mycobacterial cells wall lipids released into hosts' plasma during infection. Referenzen [1] Graessler, J.; Schwudke, D.; Schwarz, P. E.; Herzog, R.; Shevchenko, A.; Bornstein, S. R. Top-down lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients. PloS one 2009, 4, e6261.;
[2] Herzog, R.; Schwudke, D.; Schuhmann, K.; Sampaio, J. L.; Bornstein, S. R.; Schroeder, M.; Shevchenko, A. A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language. Genome biology 2011, 12, R8.; [3] Sartain, M. J.; Dick, D. L.; Rithner, C. D.; Crick, D. C.; Belisle, J. T. Lipidomic analyses of Mycobacterium tuberculosis based on accurate mass measurements and the novel "Mtb LipidDB". Journal of lipid research 2011, 52, 861-72.; [4] Zehethofer, N.; Bermbach, S.; Hagner, S.; Garn, H.; Müller, J.; Goldmann, T.; Lindner, B.; Schwudke, D.; König, P. Lipid Analysis of Airway Epithelial Cells for Studying Respiratory Diseases. Chromatographia 2014, DOI 10.1007/s10337-014-2787-5 .
A simple MALDI MS-based method to identify ether lipids in complex lipid mixtures of spermatozoa Fuchs, Beate (1); Müller, Karin (2); Schiller, Jürgen (1) 1: University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Härtelstr. 16-18, D-04107 Leipzig; 2: Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, D-10315 Berlin Stichworte: MALDI-TOF MS, ether phospholipids, phospholipase A2, 2, 4-dinitrophenylhydrazine, plasmalogens Einleitung There is increasing evidence that MALDI MS is a useful method of lipid analysis [1]: MALDI MS is fast, sensitive, and tolerates sample impurities to a considerable extent [2]. As phospholipase A2 (PLA2) cleaves selectively the sn-2 acyl group of phospholipids, PLA2 is very helpful to differentiate the residues in sn-1 and sn-2 positions. Furthermore, the classical reagent 2,4-dinitrophenylhydrazine (DNPH) is a "reactive" matrix that helps to convert aldehydes into stable products. We will demonstrate a simple approach to monitor the presence of plasmalogens (alkenyl-ether phosholipids) in spermatozoa extracts. It will also be shown that DNPH is a useful agent to derivatize selected oxidation products of lipids that are generated by the cleavage of the olefinic residues. Experimenteller Teil All chemicals, solvents, PLA2 and the applied MALDI matrices (9-aminoacridine (9-AA) and 2,5-dihydroxybenzoic acid (DHB)) and a 0.2 M DNPH solution (in phosphoric acid) were obtained from Sigma-Aldrich. Phospholipid standards were from AVANTI Polar Lipids and used as supplied. TLC/MALDI analysis was performed as recently described [3,4]. DHB was either used as 0.5 M solution in methanol or as a solution of 100 mg/ml in acetonitrile/water (1:1, v/v), while 9-AA was used in a concentration of 10 mg/ml in isopropanol/acetonitrile (60/40, v/v)) [5]. Lipid extracts from spermatozoa were obtained according to the Bligh & Dyer method and the spectra recorded as previously described [1]. Derivatization with DNPH was performed either in solution or directly on a TLC plate. Ergebnisse Spermatozoa contain in addition to common diacyl (phospho)lipids also significant amounts of ether lipids. Alkenylether lipids (plasmalogens) are of particular interest because they are considered as natural antioxidants: the alkenylether linkage has a much higher reactivity with reactive oxygen species (ROS) than the olefinic residues within the fatty acyl chains. In combination with a complex fatty acyl pattern, the unequivocal identification of plasmalogens is a challenging task - in particular if only a simple TOF device without MS/MS capacity is available. Fortunately, plasmalogens can be easily identified by combining enzymatic digestion/chemical derivatization and without the need of sophisticated MS equipment: (1) PLA2 digestion converts the plasmalogen into a lyso-plasmalogen lacking the sn-2 acyl moiety and (2) already traces of acids convert the (acid sensitive) plasmalogen into a lysolipid (lacking the ether residue) and the corresponding aldehyde. The lysolipid can be easily identified while the simultaneously generated aldehyde is not detectable under conditions of high vacuum. We will show that the derivatization of the generated aldehydes by DNPH is a convenient method to overcome this problem. The derivatization products are best detected as negative ions and this helps to minimize interferences with bulk phospholipids such as PC. Samples of boar spermatozoa will be used to illustrate the power of this strategy. Of course, the DNPH derivatization may be also performed directly on a TLC plate and gives ‑ subsequent to MALDI MS ‑ direct information about the contribution of plasmalogens to the individual spermatozoa lipid classes. Finally, it will be shown that the applied highly acidic conditions are very helpful to overcome overlap problems between different adducts and lipids with different fatty acyl compositions. It is concluded that this is a very simple and convenient approach that can be used on all MALDI mass spectrometers. Neuer Aspekte A simple method to identify plasmalogens in spermatozoa lipids was developed. This approach is also applicable to other physiologically-relevant samples.
Referenzen [1] Fuchs B, Süss R, Schiller J (2010) Prog Lipid Res 49, 450-75; [2] Schiller J, Süss R, Petković M, Hilbert N, Müller M, Zschörnig O, Arnhold J, Arnold K (2001) Chem Phys Lipids 113, 123-31; [3] Fuchs B, Schiller J, Süss R, Zscharnack M, Bader A, Müller P, Schürenberg M, Becker M, Suckau D (2008) Anal Bioanal Chem 392, 849-60; [4] Fuchs B (2012) J Chromatogr A 1259, 62-73; [5] Sun G, Yang K, Zhao Z, Guan S, Han X, Gross RW (2008) Anal Chem 80, 7576-85
LPPdb: a new MS-centered database of modified lipids Ni, Zhixu (1,2); Milic, Ivana (1,2); Weber, Dieter (1,2); Annibal, Andrea (1,2); Hoffmann, Ralf (1,2); Fedorova, Maria (1,2) 1: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig,; 2: Center for Biotechnology and Biomedicine, Universität Leipzig. Stichworte: lipidomics, lipid peroxidation, database, data management, lipid profiling Einleitung Lipid peroxidation products (LPP) generated during oxidative stress are recognized as important biomarkers of numerous human disorders. LPP are extremely diverse in their structures and thus require sophisticated detection protocols. In recent years, we established several strategies combining TLC and HPLC with MALDI- and ESI-MS to analyze LPP, such as carbonylated, hydroxylated, and head group modified phospholipids, oxysterols, eicosanoids, oxidized and nitrated fatty acids. These strategies allowed us to identify many new LPP that have not been reported before. As the currently available lipid databases, such as LIPIDMAPS, LipidHome, and HMDB, do not specifically collect information on modified lipids, we initiated a knowledge MS-centric database of oxidized lipids termed LPPdb. Experimenteller Teil LPPdb consists of two main components: LPPdb client and LPPcloud website (www.uni-leipzig.de/~oxlpp). The webclient structure was designed using python language [1] with Pandas data analysis library [2] for advanced data processing and PySide [3] for client interface. The LPP entries are parsed and stored externally in MariaDB database server [4] by LPPdb client: chemical formula, exact mass, m/z, taxonomy, and IUPAC name, structure, MS/MS information (fragment ions, neutral losses and MRM transitions), identification/quantification protocols (LC and MS specific conditions), known pathways, and reference DOI. Derivatization reagent specific information is also supported. Unique LPPid are generated automatically based on the key properties of each entry. Ergebnisse LPPdb is maintained as a crowd-sourcing database, which allows all users to generate their private LPP databases and to exchange information with others. The LPPdb client is a fundamental client software, which was designed to generate and manage all LPP database entries, whereas LPPcloud provides an online web service with a query function of a public LPP database contributed by the LPPdb community. The LPPdb client contains five modules, which cover the whole workflow including entry generation, method management, database management, database query, and entry viewer. The architecture of LPPdb allows a flexible use of private and LPPcloud databases. A preliminary dataset obtained from MS data can be used for advanced LPPdb searches to generate a list of identified LPP supported by the corresponding tandem mass spectra from the private database, collaborators database and LPPcloud to accelerate data analysis of complex biological samples.Confirmed entries can be exported in “LPP.txt” and further used as supplementary files for publications. Thus a new strategy to manage and analyze mass spectra of LPP was established. The current version of LPPdb contains general, MS related and published information to assist MS/MS interpretations. So far more than 400 entries have been generated including oxysterols, nitrated and oxidized fatty acids, eicosanoids, hydroxylated, truncated, glycated, and head group modified phospholipids, carbonylated low and high molecular weight LPP from different phospholipid classes. Researches working on oxidized lipids can get access to LPPdb and use it for the identification of oxidized lipids and for the enrichment with newly identified LPP. Neuer Aspekte The LPPdb provides a web-client based database to organize and share MS-based lipidomics data for oxidized lipids. Referenzen [1] https://www.python.org/;
[2] http://pandas.pydata.org/; [3] http://qt-project.org/wiki/PySide; [4] https://mariadb.org/
Shotgun Lipidomics for Dissecting Lipid-protein Assemblies Ayciriex, Sophie (1); Gerber, Hermeto (2); Fraering, Patrick (2); Shevchenko, Andrej (1) 1: Max Planck Institute of Molecular Cell Biology and Genetics, Deutschland; 2: Ecole Polytechnique Fédérale de Lausanne (EPFL), Brain Mind Institute and School of Life Sciences, Switzerland Stichworte: ɣ-secretase, shotgun lipidomics, lipid-protein assemblies Einleitung We combined immunoaffinity purification and shotgun lipidomics to determine the lipidome associated with gammasecretase, a multi-subunit membrane protein complex. ɣ-Secretase complex is a protease that catalyzes the final intramembrane cleavage of the -amyloid precursor protein during the neuronal production of the amyloid -peptides, the causative agents of Alzheimer’s disease (AD). It has become an important therapeutic target for the development of drugs to slow down or prevent the pathogenesis of AD. Experimenteller Teil Whole membrane lysates were prepared from CHO cells stably overexpressing the human -secretase complex according to [1]. After ultracentrifugation step, the membrane protein lysate was incubated overnight at 4°C with anti-Flag Mβ affinity beads. The -secretase complex was purified by gel filtration chromatography. Lipids were extracted using a modified Folch protocol from each collected fraction. Cholesterol was derivatized with acetylchloride prior to the analysis [2]. Lipid extracts spiked with internal standards were infused into a Q Exactive mass spectrometer (Thermo Fisher Scientific) via a robotic nanoflow electrospray ion source (Triversa NanoMate, Advion BioSciences). Lipids were identified by LipidXplorer software [3]. Ergebnisse We applied top-down shotgun lipidomics approach to quantify 53 lipid species from 11 major classes associated with affinity-purified ɣ-secretase complex with a ratio of lipids to proteins of 393. PC, PE, PS, PI and cholesterol were the major lipid classes associated with the complex, while SM, ether lipids, lysolipids and PG were detected in lesser amounts, consistently with previous studies [4]. Glycerophospholipidome was dominated by dioleoyl PC 18:1/18:1, PE 18:1/18:1, PI 18:1/18:1 and monooleoyl PS 18:0/18:1 species. Glycerophospholipids were enriched with medium chain length moieties: 30% of C16 and 60% of C18 with mainly one unsaturation (74%), two unsaturations (3%) and no unsaturation (23%). Cholesterol is a major component of lipid bilayer membrane. Some studies described cholesterol as a strong interactor of the -secretase complex. In our study, cholesterol represented 7% of the total lipid class associated with the ɣ-secretase (ratio cholesterol/protein= 30). We also observed a strong affinity between cholesterol and the complex after a second size exclusion step of the fraction enriched in affinity-purified enzyme. In this study, we described the direct lipid environment of the gamma-secretase complex by a top-down shotgun lipidomics approach. We identified the main lipid classes which could stabilize the structure and have a direct influence on the function of the membrane enzyme.
Neuer Aspekte Endogenous lipids associated with the active ɣ-secretase complex quantified by top-down shotgun lipidomics. Referenzen [1] Alattia JR, Matasci M, Dimitrov M, Aeschbach L, Balasubramanian S, et al. (2013) Highly efficient production of the Alzheimer's gamma-secretase integral membrane protease complex by a multi-gene stable integration approach. Biotechnol Bioeng 110: 1995-200; [2] Liebisch G, Binder M, Schifferer R, Langmann T, Schulz B, et al. (2006) High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim Biophys Acta 1761: 121-128.;
[3] Herzog R, Schwudke D, Schuhmann K, Sampaio JL, Bornstein SR, et al. (2011) A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language. Genome Biol 12: R8.; [4] Holmes O, Paturi S, Ye W, Wolfe MS, Selkoe DJ (2012) Effects of membrane lipids on the activity and processivity of purified gamma-secretase. Biochemistry 51: 3565-3575.
LC-MS2 based Lipid Mediator Profiling reveals insights into signaling cascades during Mycobacterium tuberculosis infection Krajewski, Matthias (1); Paudyal, Bhesh (2); Schaible, Ulrich (2); Schwudke, Dominik (1) 1: Forschungszentrum Borstel, Deutschland, Bioanalytische Chemie; 2: Forschungszentrum Borstel, Deutschland, Zelluläre Mikrobiologie Stichworte: lipid mediators, pathogen-induced inflammation, plasma, lung homogenate, LC-MS2 Einleitung Lipid mediators are bioactive derivatives of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs). They play crucial roles in the signalling network during microbial infections and mark the inflammation status of the host [1]. Lipid mediators such as prostaglandins and leukotrienes act in a pro-inflammatory manner, whereas resolvins and maresins have antiinflammatory and pro-resolving properties, respectively [2]. Both mediator types exert their regulatory effects by binding to G protein-coupled receptors that mediate neutrophil influx and macrophage stimulation [3]. Only the quantitation of these constitutional isomers using LC-MS2 approaches [4] enables a comprehensive insight into the regulation of immune cell activities. Experimenteller Teil Wild type (WT) mouse strain (C57BL/6) was used to perform a time course experiment applying aerosol infection with Mycobacterium tuberculosis (M.tb.). During 100 days of infection WT plasma (150 µl) and lung homogenate (1.0 ml) were analysed for lipid mediators. After Bligh&Dyer extraction [5] total lipids were loaded solid phase extraction (SPE)-free on a Phenomenex™ Luna C18(2) column for LC separation (Agilent 1100). Acidified mobile phase (flow rate: 10 µl/min) comprised water, acetonitrile, ammonium acetate, methanol, methyl tert-butyl ether, isopropanol. Full MS2 spectra were acquired in the negative ion mode using a Q-TOF Ultima™ equipped with an electrospray ion source. Ergebnisse LC-MS2 analysis enabled the assignment of 28 lipid mediators of our pool of standards. In this manner, the developed LC gradient allowed SPE-free loading of total lipid extracts on the column. Remaining phospholipids on column could be removed largely during a chromatographic run. The 5 deuterium-labeled internal standards for quantitation, PGE2-d9, RvD2-d5, LTB4-d4, 5-HETE-d8 and AA-d11, co-eluted with their respective endogenous species. Selected reaction monitoring of the 23 reference compounds showed a linear response in a range of 50-2500 fmol on column (r2 > 0.99). The limit of detections was determined to be 50 fmol for PGE2-d9, 62.5 fmol for RvD2-d5 and 156 fmol for 5-HETE-d8 on column when 250 µl growth medium (DMEM, containing 10% FCS) was processed as matrix. Extraction efficiencies were 104 ± 15 % (PGE2-d9), 105 ± 13 % (RvD2-d5) and 110 ± 14 % (5-HETE-d8). We quantified 28 molecular species in plasma and 36 in lung homogenate during M.tb. infection. M.tb. seemed to perturbate the host’s oxidized PUFA metabolism. When M.tb infection were studied in a time course extensive changes in the lipid mediator profiles were observed. The M.tb. infection changed the pool of di-hydroxylated PUFAs and prostaglandins. Several metabolites are present only upon M.tb. infection. 9 lipid mediators were changed significantly (5: increased; 4: decreased) in plasma compared to the uninfected WT control, among them the proinflammatory PGE2, the bioactive 12-HETE and the pathway marker 14-HDoHE. The pro-inflammatory LTB4 was detected only at day 30 after M.tb. infection. Our pilot experiments might help to understand the specificity of lipid signalling during M.tb infection. Therefore, in our future work we will apply the LC-MS2 platform for a systematic analysis of infection-induced inflammation specific to selected pathogens. Neuer Aspekte A SPE-free LC-MS2 method to read out on lipid mediator level the inflammation status of the host upon infection. Referenzen [1] TAM, V. C. 2013. Lipidomic profiling of bioactive lipids by mass spectrometry during microbial infections. Semin Immunol, 25, 240-8.;
[2] SERHAN, C. N., DALLI, J., COLAS, R. A., WINKLER, J. W. & CHIANG, N. 2014. Protectins and maresins: New pro-resolving families of mediators in acute inflammation and resolution bioactive metabolome. Biochim Biophys Acta.; [3] BUCKLEY, C. D., GILROY, D. W. & SERHAN, C. N. 2014. Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity, 40, 315-27.; [4] LE FAOUDER, P., BAILLIF, V., SPREADBURY, I., MOTTA, J. P., ROUSSET, P., CHENE, G., GUIGNE, C., TERCE, F., VANNER, S., VERGNOLLE, N., BERTRAND-MICHEL, J., DUBOURDEAU, M. & CENAC, N. 2013. LC-MS/MS method for rapid and concomitant quantification of pro-infl; [5] LIU, X., MOON, S. H., MANCUSO, D. J., JENKINS, C. M., GUAN, S., SIMS, H. F. & GROSS, R. W. 2013. Oxidized fatty acid analysis by charge-switch derivatization, selected reaction monitoring, and accurate mass quantitation. Anal Biochem, 442, 40-50.
Eliminating systematic errors in the LC-MS/MS analysis of vitamin D in human serum Volmer, Dietrich; Qi, Yulin Universität des Saarlandes, Deutschland Stichworte: Vitamin D, LC-MS/MS, Ion mobility, Systematic errors, Accuracy Einleitung LC-MS/MS determination of concentration levels of 25-hydroxyvitamin D in human serum is difficult for a number of reasons, including the low ionization efficiency and problems with selectivity. The latter problems are only partially addressed through stable isotope internal and reference standards. Many issues of method selectivity remain unresolved, because isobaric ions have the potential to interfere in, even in MS/MS mode. This study was concerned with systematic errors and accuracy of LC-MS/MS assays for vitamin D in human serum and suggests an alternative solution for avoiding/minimizing these interferences. For this, we compared different sample preparation techniques and utilized a combination of low-resolution triple quadrupole, ultra-high resolution Fourier transform ion cyclotron MS and differential ion mobility spectrometry. Experimenteller Teil After initial protein precipitation, human serum samples were extracted using liquid/liquid, solid-phase or plate microextraction. Separation of vitamin D metabolites was achieved using a PFP column and compounds ionized by electrospray ionization. Low resolution tandem MS was conducted on a quadrupole-quadrupole linear ion trap instrument (QqLIT) in multiple reaction monitoring (MRM) mode; ultra-high resolution characterization experiments on a 7 Tesla quadrupole-Fourier-transform ion cyclotron resonance (qFTICR) MS. Overlap of peaks was simulated using MatLab; the Gaussian function was used to reconstruct the peak shape and peak areas. Differential ion mobility (DMS) separations were performed between ESI source and entrance quadrupole q 0 of the QqLIT instrument. No chemical modifier was added to enhance DMS separations, to improve ruggedness of the method. Ergebnisse In this study, we have systematically investigated the range of isobaric compounds present in human serum for the main vitamin D metabolite, 25-hydroxyvitamin D ([M+H]+, m/z 401), which is commonly measured to determine vitamin D status of individuals. We were specifically interested in those compounds that have the potential for causing interferences in MS/MS as a result of common product ions. During the experiments, we varied the sample preparation technique for extraction of 25-hydroxyvitamin D from serum, to compare their impact on the range of coextracted isobaric interferences during electrospray ionization. We have shown by detailed high resolution FTICR mass spectrometry experiments that multiple isobaric compounds were present in the sample extracts from human serum, several of which influenced peak areas measured for 25-hydroxyvitamin D using the LC-MS/MS assay in multiple reaction monitoring (MRM) mode. At least one of these isobars was an exogenous compound, the structure of which was fully elucidated, as will be shown in the presentation. We believe that the interference was probably introduced by our analytical methodology; other interferences were endogenous molecules from the serum matrix. We have also evaluated differential ion mobility spectrometry as a filtering step, to successfully remove or reduce the level of the isobaric interferences. Our experiments demonstrated that under the specific experimental conditions chosen in our assay, up to 20% of the vitamin D signal originated from interfering components in the sample extracts, which were indistinguishable from vitamin D using the regular assay, even under MS/MS conditions. We therefore believe that differential ion mobility has the potential to significantly decrease systematic errors, and thus improve the accuracy of vitamin D assays. Neuer Aspekte Detailed experimental investigation of the ’isobaric space’ of interferences of vitamin D from endogenous and exogenous compounds in human serum
The new Agilent IMS QTOF, how it works and where it does add advantages Joachim, Thiemann; Falter, Ralf Agilent Technologie, Deutschland Stichworte: Ion Mobility, Native MS, Metabolomics, CCS Einleitung Ion mobility spectrometry (IMS) is a gas phase ion separation technique that can be coupled to mass spectrometry. The separation is carried out in a drift tube filled with an inert buffer gas and the ions are transmitted across the drift tube using an electric field gradient. Larger ions traverse the drift tube at a slower pace than the smaller ions due to their larger collision cross section, thus providing the basis for ion separation. The drift time information obtained using this technique allows the user to directly calculate the collision cross section (CCS) for ions of interest. The CCS information is related to the structure of ions and it can be used in biological, structural, kinetic and thermodynamic studies of molecules. Experimenteller Teil In this presentation we will present a new uniform-field ion mobility-quadrupole time-of-flight mass spectrometer (IMS-QTOF-MS). The uniform low field drift cell is independent in its resolution from RF voltages and therefor effects from ion heating can be separately controlled and is overall very low. The design of the ion optics with the drift cell after the entrance funnels and before the the quadrupol enables us to have constant drop in pressure downstream the ion optics, thus grantees the structural integrity of all kinds of analysts. Ergebnisse The application examples for Metabolites, Carbohydrates, Peptides and native Protein (complexes) will illustrate, that the system add great advantages to dedicated applications. On top on the superior sensitivity of the systems, it does provide drift separation and CCS information which can be used for identification or confirmation of compounds without any intensive drift tube calibration. We will demonstrate the sensitivity and separation of isobaric compounds with an urine sample. Proofing the concept of the system, we can show that more compounds can be found with the IMS system, than with a non IMS system of same performance class. We can also demonstrate that a non IMS system can lead to wrong relative Quant information. Carbohydrates do show a great complexity by linkage. Ion mobility is very often the only possible TOF coupled technology to separate these type of molecules which we be shown on one example. We have also evaluated the Agilent low field drift tube ion mobility device for IM native MS applications, using Nano electrospray ionization. CCS values for Concanavalin A and Cytochrome C are shown. The data will illustrate that for Con A with a charge state of 20, several conformers with CCS values lower than the literature values can be found. Neuer Aspekte Particularly easy and accurate determination of the CCS of compounds. High IMS resolution for separation of isobaric compounds.
Weiterführende Untersuchungen zum Vergleich von APLI und APCI bei der Anwendung in der Ionenmobilitätsspektrometrie Ihlenborg, Marvin; Raupers, Björn; Grotemeyer, Jürgen Christian-Albrechts-Universität zu Kiel, Deutschland Stichworte: APCI, APLI, Atmosphärendruck IMS Einleitung Nach ersten erfolgreichen Vergleichsmessungen von APCI und APLI Quellen an einem selbstgebauten IMS bei Atmosphärendruck, sollen weiterführende Vergleichsmessungen an einem kommerziell verfügbaren IMS der Firma Dräger durchgeführt werden. Durch die Verwendung eines kommerziellen Aufbaus soll eine größere Vergleichbarkeit der erhaltenen Driftzeitspektren mit der Literatur erhalten werden. Es wird zum jetzigen Zeitpunkt davon ausgegangen, dass eine starke Signalverbreiterung bezogen auf die Dauer der Ionisation erhalten wird. Experimenteller Teil Ein kommerziell erworbenes IMS der Firma Dräger (Dräger Safety, Dräger IMS 5000) wurde für den Betrieb unter APCI Bedingungen und einer abwechselnden Messung unter APLI Bedingungen umgebaut. Zur Ionisation unter APCI Bedingungen steht eine 3H-Quelle zur Verfügung. Für APLI Untersuchungen wird ein Nd:YAG Laser (Surelite I, Continuum, 4. Harmonische, 266 nm) verwendet. Ergebnisse Durch die Änderung der Ionisationsmethode bei ansonsten nahezu vergleichbaren Bedingungen können mehrere Effekte in den erhaltenen Driftzeitspektren beobachtet werden. Neben der möglichen Ionisation von kleinen aromatischen Verbindungen mit einer Polarität kleiner als Wasser, werden bei den APLI Untersuchungen für alle hier untersuchten Substanzen bereits bei geringen Laserleistungen höhere Ionenausbeuten im Vergleich zu APCI erreicht. Auffällig bleibt jedoch eine annähernd vergleichbare Halbwertsbreite der Signale. Durch sehr kurze Ionisationsdauern durch einen Laserpuls (~ 10 ns) im Vergleich zum Extraktionspuls bei APCI (~ 110 µs) wäre eine entsprechende Verringerung der Halbwertsbreite zu erwarten. Mehrere der folgenden Effekte sind vorstellbar. Es werden Fragmente mit unterschiedlichen Driftzeiten auf Grund eines Leiter-Wechsel-Mechanismus und durch Ionen-Molekülreaktionen mit dem Probengas und Driftgas erhalten. Auch Cluster-Ionen unterschiedlicher Zusammensetzung wären so denkbar. Als Folge von zeitlicher, energetischer und räumlicher Verteilung der Moleküle vor dem Repeller werden zusätzlich langsamere Ionen erzeugt. Der Energieübertrag der Photonen auf die Moleküle ist so groß, dass eine explosionsartige Verteilung der Ionen in alle Raumrichtungen mit großer kinetischer Energie stattfindet. Neuer Aspekte Qualitative und quantitative Vergleichbarkeit von Ionenausbeuten und Driftzeitspektren erhalten durch APCI und APLI mit 266 nm.
Gas-Phase Microsolvation of Ubiquitin: Identification of Crown Ether Binding Sites Göth, Melanie (1); Schmitt, Xiao Jakob (2); Warnke, Stephan (2); von Helden, Gert (2); Pagel, Kevin (1,2) 1: Freie Universität Berlin, Institute of Chemistry and Biochemistry, Takustraße 3, 14195 Berlin, Germany; 2: Fritz Haber Institute of the Max Planck Society, Department of Molecular Physics, Faradayweg 4-6, 14195 Berlin, Germany Stichworte: ion mobility-mass spectrometry, microsolvation, ubiquitin, crown ether, gas-phase structure Einleitung There is ongoing debate about the extent to which protein structure is retained after electrospray ionization and transfer into the gas phase. It is widely accepted that the repulsion between equal charges is one of the major determinants: low charge states typically adopt compact, native-like conformations, while high charge states tend to unfold. Intermediate charge states can show a multitude of coexisting conformations.[1] Recently, we showed that not only Coulomb repulsions but also the much more subtle coordination of charged side chains onto the protein backbone can severely influence the conformation of gas-phase proteins.[2] Here, we are investigating these effects further by comparing the impact of solvent-exposed charged residues with those involved in intramolecular salt bridges. Experimenteller Teil In order to study the influence of side chain-backbone coordination on the structure of a gas-phase protein, we noncovalently attached different amounts of crown ether (CE, 18-crown-6) to the charged lysine side chains of a series of ubiquitin mutants (Sigma Aldrich). The resulting protein-CE complexes were analyzed via tandem MS and ion mobility-mass spectrometry (IM-MS) using a commercially available traveling wave IM-MS instrument (Waters, Synapt G2-S). Ergebnisse 18-crown-6 is known to strongly bind to protonated lysine and, with considerably lower affinity, arginine side chains in gas-phase proteins. In order to study the impact of such CE microsolvation on the protein structure, we here investigated ubiquitin mutants, in which either all lysine residues (noK), or all but one specific lysine (K6, K11, K27) are replaced by arginine. Independent of the charge state, complexes with up to five CEs were observed for wt ubiquitin as well as all mutants at gentle conditions. CID experiments revealed, however, that there are significant differences in the relative dissociation energy between lysine and arginine-bound CE. IM-MS experiments showed that ubiquitin ions of high (unfolded) and low charge states (compact) essentially retain their shape upon CE complexation. At intermediate charge states on the other hand, wt ubiquitin undergoes an unusually distinct compaction with increasing number of attached CEs. This is in agreement with previous observations [2] and can be explained by the fact that the CE solvates the protonated lysine side chains and prevents them from a structure-breaking coordination to the protein backbone. In contrast to wt ubiquitin, no significant structural compaction upon CE binding was observed for the noK mutant, which likely results from the weak binding of CE to arginine. A similar trend was also observed for the K11 and K27 mutants, in which the only remaining lysine is involved in a strong intramolecular salt bridge. Surprisingly, however, a clear compaction was found for K6 CE complexes, in which the lysine side chain is solvent-exposed and easily accessible for complexation. Taken together, our results indicate that the unfolding induced by coordination of charged side chains to the protein backbone strongly depends on the involved residues. Solvent-exposed lysines exhibit a much stronger impact on the protein structure than those buried in salt bridges.
Neuer Aspekte Identifying crown ether binding sites with ion mobility-mass spectrometry using different ubiquitin mutants. Referenzen
[1] Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F.; J. Am. Chem. Soc. 1997, 119, 2240-2248.; [2] Warnke, S.; von Helden, G.; Pagel, K.; J. Am. Chem. Soc. 2013, 135, 1177-1180.
Analysis of isomeric lipids by high resolution ion mobility-mass spectrometry Größl, Michael (1); Graf, Stephan (1); Lisa, Miroslav (2); Holcapek, Michal (2); Sampaio, Julio (3); Dick, Bernhard (4); Vogt, Bruno (4); Knochenmuss, Richard (1) 1: Tofwerk, Schweiz; 2: University of Pardubice, Czech Republic; 3: Lipotype, Germany; 4: Bern University Hospital, Switzerland Stichworte: Isomer separation, ion mobility, lipids, steroids, sterols Einleitung Ion mobility spectrometry coupled to mass spectrometry (IMS-MS) is becoming increasingly popular, but most commercial systems have insufficient ion mobility resolution to separate isomeric compounds. We report on a benchtop IMS-MS that delivers resolution beyond 200, under true low field conditions, with 50% duty cycle. The benefits of this system coupled to TOF-MS are demonstrated for the analysis of isomeric biomolecules. Experimenteller Teil All measurements were carried out on a Tofwerk IMS-TOF. The systems comprises an ESI source, a 10 cm desolvation tube, a 20 cm drift tube (both made from resistive glass) and a Tofwerk HTOF TOF-MS. Measurements were carried out in both positive and negative ion modes (applied ESI potential approximately 2kV) with the desolvation and drift tube kept at pressures between 1 and 1.4 bar and 150°C with nitrogen as the buffer gas. The reduced field strengths were near 2 Td.
Ergebnisse IMS separates molecules based on their collision cross sections. Since this depends on the geometry of molecules, IMS is an attractive method for isomer separation. In most cases resolving power above 100 is required to distinguish isomers, a range which lies beyond most instruments. Here we show separation of isomeric biomolecules in a gas-tight multiplexed IMS drift cell at pressures of up to 1.4 bar. Combined with multiplexing postprocessing techniques, ion mobility resolving power above 200 was obtained. Multiplexing also increases ion transmission over 200 times and S/N ratios 10 times compared to conventional pulsed mode. For phospholipids and triacylglycerols, we show the separation of regioisomers (different positions of acyl chains on the glycerol skeleton), double bond positional isomers and stereoisomers (double bond geometry). We also show that, based on accurate reduced mobility measurements, identification of lipid species is feasible even in complex samples such as total lipid extracts. Additionally, high resolution IMS-MS was investigated for the analysis of closely related isomeric steroids and their metabolites which are commonly included in clinical urine steroid profiles. We demonstrate that the technique is able to separate isomers of hydroxyprogesterones or cortisone metabolites. Finally, we also present data that highlights separation of isomeric sterols (cholesterol derivatives). Neuer Aspekte Analysis of isomeric lipids, steroids and sterols by ion mobility-mass spectrometry.
Altersbestimmung von Kugelschreibereinträgen durch Lösungsmittel, Harze und Binder. Hahn, Andrea (1); Seiler, Peter (1); Köhler, Fritz (1); Kirsch, Dieter (2) 1: BKA, Kriminaltechnisches Institut, KT 41 Kriminaltechnisches Institut, KT 12 Zentrale Analytik II
Physikalisch-chemische
Urkundenprüfung;
2:
BKA,
Stichworte: Altersbestimmung, Kugelschreiber, Polymeranalytik Einleitung In Kugelschreibereintragungen finden mit der Zeit chemische Veränderungen der verschiedenen Komponenten statt.[1] Diese chemischen Veränderungen können zur Altersbestimmung der Eintragung und somit des Vertrags oder Dokuments verwendet werden. Zu den Komponenten in den Kugelschreiberpasten gehören Lösungsmittel, Farbstoffe, Harze, Binder und weitere Additive. In den letzten Jahren wurden verschiedene analytische Methoden zur Altersbestimmung von Kugelschreibereintragungen entwickelt, die auf den Lösungsmitteln in den Pasten basieren. Das KTI entwickelte hierzu eine HPLC-UV Methode, mit der eine Alterungsbestimmung über das Lösungsmittel Phenoxyethanol durchgeführt wird.[2] Diese Methode wird bereits in der Fallarbeit eingesetzt und ermöglicht die Altersbestimmung einer Kugelschreibereintragung bis zu einem Zeitraum von 6 Monaten.
Experimenteller Teil Zur Erweiterung des Datierungszeitraums wird auch das Alterungsverhalten von Harzen, Bindern und Additiven in Kugelschreibereintragungen untersucht. In den letzten Monaten wurde im Zuge des Monopoly Projektes 2011 eine massenspektrometrische Methode entwickelt, mit der Harze, Bindemittel und Additive sensitiv detektiert werden. Dazu gehören Probenaufbereitung, HPLC und hochauflösende Massenspektrometrie. Verschiedene Ionisationsmethoden, wie die Electrospray Ionization (ESI), die Atmospheric Pressure Chemical Ionization (APCI) und die Direct Analysis in Real Time (DART) wurden verwendet. Für einen großen Anteil der Analyte ist die positive ESI die geeignetste Ionisierungmethode. Ergebnisse Verschiedene chemische Komponenten von Kugelschreibertinten wurden massenspektrometrisch analysiert, charakterisiert und die Massenspektren und Fragmentspektren werden in einer Datenbank zusammengefasst. Eine qualitative Unterscheidung zwischen verschiedenen Pasten und Eintragungen ist bereits möglich. Analysen von gealterten Proben zeigen, dass Harze, Binder und Additive sehr langsam altern. Sowohl die Ausgangsverbindungen wie auch die Abbauprodukte sind über einen langen Zeitraum von mehreren Jahren bis Jahrzehnten in Kugelschreibereintragungen detektierbar. Neuer Aspekte Analytik von Harzen, Bindemitteln und Kugelschreibereinträgen über mehrere Jahre.
Additiven
in
Schreibmitteln
und
Altersbestimmung
von
Referenzen [1] [1] M. Ezcurra et al. "Analytical methods for dating modern writing instrument inks on paper." Forensic Science International 197.1 (2010): 1-20.; [2] [2] J. Bügler, H. Buchner, and A. Dallmayer. "Age Determination of Ballpoint Pen Ink by Thermal Desorption and Gas Chromatography-Mass Spectrometry." Journal of Forensic Sciences 53.4
Structural investigation of co-assembled clathrin adaptor protein complexes Heidemann, Johannes (1); Garcia-Alai, Maria Marta (2); Gieras, Anna (2); Meijers, Rob (2); Uetrecht, Charlotte (1) 1: Heinrich-Pette-Institut, Deutschland; 2: EMBL Hamburg Unit, European Molecular Biology Laboratory (EMBL), Hamburg, Deutschland Stichworte: Native Mass Spectrometry, Endocytosis, Protein complexes, Lipid-binding Einleitung Clathrin-mediated endocytosis is the major trafficking route from the plasma membrane to the cytoplasm and thus indispensable for many cellular processes. The remodeling of the membrane during endocytosis requires a mechanical force to be transmitted to the membrane. In this context, clathrin adaptors were found to have two important functions. They link clathrin to the plasma membrane and also couple the membrane to the polymerizing actin cytoskeleton. Experimenteller Teil Lipid binding of single membrane-binding domains of clathrin adaptors from yeast and fungi was analyzed. Furthermore, complex stoichiometry and clues on the complex topology were obtained. Ergebnisse Here, we show that the membrane-binding domains of two clathrin adaptors co-assemble in a lipid-dependent manner and obtain structural information of the protein complex by native mass spectrometry. Neuer Aspekte Structural information on co-assembled clathrin adaptor protein complexes
At the edge of lipidOMICS and proteOMICS: mobility mass spectrometry of lipid-protein adducts Fedorova, Maria (1,2); Milic, Ivana (1,2); Ni, Zhixu (1,2); Griesser, Eva (1,2); Hoffmann, Ralf (1,2) 1: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig; 2: Center for Biotechnology and Biomedicine, Universität Leipzig Stichworte: Lipidomics, lipid peroxidation, lipid-protein adducts, LC-MS, ion mobility, proteomics Einleitung Many human diseases are accompanied by chronic inflammation and closely connected to oxidative stress (OS). OS can oxidize virtually all biomolecules including lipid peroxidation generating multiple electrophilic products capable to modify nucleophilic sites in proteins. The heterogeneous nature of lipid peroxidation products (LPP) and the large variety of LPP-protein adducts demands sophisticated strategies to identify such protein modifications by mass spectrometry, which starts with a detailed profiling of reactive LPP produced under OS. The combination of the lipidomics data provide the basis for a proteome-wide analysis of LPP-peptide adducts. Here we present lipidomics of OS-derived LPP combined with proteomics of modified proteins for high-throughput identification of lipid-protein adducts both in vitro and in vivo. Experimenteller Teil The analysis of the LPP relied on their specific derivatisation followed by LC-MS 1,2 to construct a database (LPPdb) of reactive LPP3, which was used for search for LPP-adducts in synthetic model peptides separated by RPC or HILIC and analyzed using high resolution ESI-Orbitrap- and ESI-IMS-MS (Synapt G2 Si). The CID spectra revealed certain rules to distinguish different types of LPP-peptide adducts. Additionally, IMS-MS allowed the separation and characterization of conformational isomers formed between peptides and phospholipid-derived LPP. This information allowed finally the in-depth profiling of complex LPP-protein adduct mixtures in cell models of oxidative stress by combining LC-MS relying on extended exclusion lists with different fragmentation techniques (CID and ETD). Ergebnisse The newly developed LC-MS protocol relying on the specific derivatisation of carbonylated LPP identified more than 150 low- and high-molecular weight reactive lipids generated by in vitro oxidation of phospholipid (PL) mixtures and complex cellular extracts. This information was used to establish an open database (termed LPPdb 3) consisting of MScentered information on oxidized lipids. Reactive LPP entries allowed the characterization of LPP-peptide adducts using different LC, MS, and IM-MS techniques. The acquired CID tandem mass spectra allowed distinguishing Schiff bases, Michael adducts, and dehydrated cyclic hemiacetals formed by LPP on Lys-, Cys-, and His-residues. IMS-MS allowed the separation of different classes of compounds (peptide vs lipid vs peptide-lipid) and various structural conformers within a single peptide-lipid adduct, driven most probably by the orientation of the hydrophobic lipid along the peptide backbone. The information obtained on the in vitro systems was the applied to a cardiomyocyte model of OS. Lipid extracts were derivatized (reactive LPP), identified, and relatively quantified by nanoRPC-ESI-MS. The 25 dominating LPP species (e.g. alkanals, alkenals, alkadienals, alkatrienals, and oxo-carboxylic acids) were dynamically generated and degraded with different kinetics. Several PL-bound aldehydes were identified for the first time after OS. Furthermore, the protein extracts were digested, analyzed by LC-MS, and the library of reactive LPP was used for identification of LPP-protein adducts. All protein modifications significantly increased during OS. The combination of lipidomics and proteomics allowed the identification of more than 200 proteins modified by LPP species of which many are involved in calcium signaling pathways, regulation of actin cytoskeleton, focal adhesion, and phosphatidylinositol signaling system. Biochemical and microscopy studies confirmed LPP-derived impairment of Ca-signaling and cytoskeletal protein distribution. Neuer Aspekte The combined lipidomics/proteomics-strategy provides powerful approach to analyze lipid-modified proteins in OSrelated diseases and cellular physiology.
Referenzen [1] I. Milic, R. Hoffmann, M. Fedorova (2013)Simultaneous detection of low and high molecular weight carbonylated compounds derived from lipid peroxidation by ESI-MS/MS. Anal Chem. 85(1): 156-162.; [2] I. Milic, M. Fedorova.(2015) Derivatization and detection of small aliphatic and lipid-bound carbonylated lipid peroxidation products by ESI-MS. Meth Mol Biol. Advanced Protocols in Oxidative Stress III. Humana Press. 1208, 3-20.; [3] www.uni-leipzig.de/~oxlpp
LUX Score: Cheminformatics Approach to Compute Lipidome Homology Marella, Chakravarthy (1); Torda, Andrew (2); Schwudke, Dominik (1,2,3) 1: Research Center Borstel, Deutschland; 2: Centre for Bioinformatics, University of Hamburg; 3: Airway Research Center North, German Center for Lung Research Stichworte: LUX Score, Cheminformatics, Lipidome Einleitung Lipids play major role in human health and disease. To understand their function, model organisms like D. melanogaster, S. cervisiae, C. elegans etc. are commonly employed. However, it is known that lipid structures in these model organisms are different from humans. To extrapolate the functional implications of model organism’s lipidome to humans, a criterion is necessary. We hypothesized that lipid structures and their structural relation to each constituent of a given lipidome might be the best link to function. Lipidome Juxtaposition Score (LUX Score) describes the-overall-structural-differences between lipids of any given pair of cells/tissues or organisms. We develop the LUX Score with the aim to provide a tool that facilitates systems biology research. Experimenteller Teil The workflow to determine LUX Score starts by combining all lipids from a set of lipidomes into a unique list. It is followed by conversion of lipid structures to SMILES. Next, we determine similarity between all pairs of SMILES from the unique list. Thereafter, we applied Principal Component Analysis (PCA) to convert pairwise structural similarity values of SMILES into three dimensional chemical space coordinates. Next, we determine the extent of overlap between a pair of lipidomes by comparing the spatial distribution their respective lipids in the chemical space. As a proof of principle we tested the accuracy of LUX Score(s) by comparing lipidomes of yeast strains that were grown at 24°C and 37°C [1]. Ergebnisse SMILES (Simplified Molecular Input Line Entry Specification) is a linear representation of three-dimensional structure of a molecule, routinely used for storing and retrieving chemical structures from databases [2]. We tested common algorithms for retrieving structures from chemical libraries. Canonical SMILES were initially developed for this purpose [3]. Canonical SMILES provide one unique representation of a given chemical structure even though, theoretically, one could write many SMILES for one structure. However, we found out that isomerism of lipids could not be represented with canonical SMILES. As result we developed template-based SMILES based on LIPIDMAPS Structure Drawing Tools [4] that keep relationships between lipid classes based on a chemical nomenclature. This approach also resulted in one unique SMILES but enabled us to utilize sequence analysis algorithms such as Bioisosteric similairty, Smith-Waterman, SMILIGN and Levenshtein distance. Among the six methods tested, Levenshtein distance provided most consistent similarity scores. We show that PCA produces unique coordinates for all 30150 lipids of the LIPIDMAPS structure database and cluster lipids according to the defined nomenclature. Plotting lipids in orthogonal principal component planes provided an intuitive visualization system for navigating within the yeast lipidome comprising 248 unique lipids. We measure the extent of overlap between a pair of lipidomes by calculating the average distance between their unique lipids in the PCA chemical space. Thus, LUX Score is a numerical measure of the combined structural differences between a pair of lipidomes. LUX Score based clustering of yeast lipidomes brought the wild-type BY4741 and mutant-Elo1 strains together, indicating that the metabolic alteration caused by the Elongase mutation was smaller than the temperature effect. For the mutant strains Elo2 and Elo3 we noticed stronger influence of the Elongase mutation, separating them from the wild-type BY4741 and mutantElo1 strains. Neuer Aspekte For the first time, a homology measure is described for lipidomes. Referenzen [1] PNAS, 2009, 106(7):2136-2141;
[2] J. Chem. Inf. Model., 1988, 28(1):31-36; [3] J. Chem. Inf. Model., 1989, 29(2):97-101; [4] J. Cheminformatics, 2012, 4(1): 23
Investigation of UV transformation products of xenobiotics in the aquatic environment by means of liquid chromatography and mass spectrometry Roscher, Jörg; Karst, Uwe Westfälische Wilhelms-Universität Münster, Deutschland Stichworte: Emerging Contaminants, UV-degradation, HPLC-MS, MS(n) Einleitung The wide use of medicines, pesticides, and personal care products leads to increasing concentrations of xenobiotics in the aquatic environment [1,2]. Since conventional waste water treatment plants degrade these contaminants insufficiently, additional processes (advanced oxidation processes, AOP) are investigated and already tested in pilot projects [3]. Several AOPs have been investigated, including ozonation, UV-radiation, Fenton’s reaction, and combinations therof. They all aim at the production of hydroxyl radicals, which undergo further reactions with the contaminants. In literature, many impressive degradation efficiencies for a wide range of common contaminants have been presented [3-5], but often only the decreasing concentration of the investigated xenobiotic is presented, without any information regarding reaction products. Experimenteller Teil Two common xenobiotics occurring in the aquatic environment are Diclofenac and Sulfamethoxazole, which are known to be easily degraded with AOPs. In this project, they were degraded by means of pure UV radiation (220-500 nm). After different radiation times, the samples were analyzed by liquid chromatography-mass spectrometry (LCMS). This way, a kinetic profile for both, the degradation process of each xenobiotic and the occurring degradation products, could be recorded. The use of a time of flight mass spectrometer (TOF-MS) allows determination of mass to charge ratios with high accuracy, so that sum formulas can be proposed. Additional fragmentation with a 3D-ion trap provides information about the fragmentation behavior and allows the prediction of tentative chemical structures. Ergebnisse Both investigated xenobiotics show a high number of products after UV radiation. For each substance, at least one occurring product is unstable against further UV radiation. Its intensity first increases to a maximum followed by a decrease of this product, which indicates its subsequent degradation. Other products appear after different radiation times and show increasing intensities until they reach a stable maximum intensity. In case of Diclofenac, the isotopic patterns of the products show that the metastable product still carries one chlorine atom, whereas for all other products, no chlorine can be found anymore. Detection by means TOF-MS reveals sum formulae for the degradation products with low mass deviations. Together with obtained fragmentation spectra, tentative structures of some of the products can be presented. Some of the detected degradation products have already been described in the literature, but the presented work includes a range of previously undescribed products, which are of particular interest for further experiments regarding their reactivity and structural characterization. Furthermore, a comparison between pure UV radiation and UV/H2O2 as another AOP is carried out to see, if the addition of hydrogen peroxide accelerates the degradation process and if it leads to the same products. Neuer Aspekte Photochemistry/LC-MS of xenobiotics allows the identification of their UV degradation pathways. Referenzen [1] B.A. Wols, C.H.M. Hofman-Caris; Review of photochemical reaction constants of organic micropollutants required for UV advanced oxidation processes in water, Water Research 2012, 46, 2815-2827.; [2] M. Gros, M. Petrovic, D. Barcelo; Analysis of Emerging Contaminants of Municipal and Industrial Origin, Handbook of Environmental Chemistry. Vol. 5S/1, 2008, 37-104.;
[3] S. Esplugas, D.M. Bila, L.G.T. Krause, M. Dezotti; Ozonation and advanced oxidation technologies to remove endocrine disrupting chemicals (EDCs) and pharmaceuticals and personal care products (PPCPs) in water effluents, J. Hazard. Mater. 2007, 149(3), 631; [4] F. Yuan,C. Hu, X. Hu,J. Qu, M. Yang; Degradation of selected pharmaceuticals in aqueous solution with UV and UV/H2O2, Water Research 2009, 43(6), 1766-1774.; [5] T.A. Ternes, J. Stüber, N. Herrmann, D. McDowell, A. Ried, M. Kampmann, B. Teiser; Ozonation: a tool for removal of pharmaceuticals, contrast media and musk fragrances from wastewater? Water Research 2003, 37, 19761982.
Electrochemistry coupled to LC/MS for the identification and characterization of reactive xenobiotic metabolites and their protein adducts Wigger, Tina (1,2); Büter, Lars (1,2); Karst, Uwe (1) 1: University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 48149 Münster, Germany; 2: NRW Graduate School of Chemistry, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany Stichworte: Metabolism, Protein adducts, Electrochemistry Einleitung Adduct formation of reactive metabolites with proteins is considered as a process contributing to the toxicity of xenobiotics. Therefore, the identification and characterization of reactive metabolites and their protein adducts is of great importance in toxicological research. Electrochemistry coupled online to liquid chromatography and mass spectrometry (EC/LC/MS) is a purely instrumental method that has proven to be a valuable tool not only for simulating the metabolic transformation reactions of xenobiotics but also for studying the generation of their protein adducts. In this presentation, we introduce the general principle and setup of this method and its application for analyzing the metabolic processes of polycyclic aromatic hydrocarbons (PAHs), a group of ubiquitous environmental contaminants. Experimenteller Teil The solution containing the analyte is pumped through a commercially available electrochemical cell, in which the molecules are oxidized on the surface of the working electrode. Generated oxidation products are either detected directly by means of mass spectrometry or after an additional separation step using an integrated HPLC system. For studying adduct formation, the setup is extended by a second syringe pump and a T-piece, which allows the addition of biomolecules into the effluent of the EC cell. Identification and characterization of generated protein adducts is achieved by LC/MS analysis of the intact proteins as well as their peptides obtained by tryptic digestion. Ergebnisse Pyrene and benzo[a]pyrene were selected as PAH model compounds and oxidized within an electrochemical thinlayer cell, equipped with a boron-doped diamond (BDD) working electrode. The generated oxidation products were analyzed using high resolution mass spectrometry (HRMS) in order to obtain their exact masses. By applying a potential ramp, mass voltammograms were recorded, which show the obtained mass spectra in dependency on the applied voltage in a three dimensional plot. These plots give a fast overview of the oxidation behavior of the analytes and may provide first information on the formation of reactive species. For both investigated model compounds, quinones were identified as the major oxidation products. Further characterization of these oxidation products was achieved by means of HPLC separation as well as MS/MS analysis. Since quinones represent a class of metabolites that generally show a high potential to form adducts, the next step was to study the reactivity of the electrochemically generated PAH metabolites towards biomolecules. For this purpose, the tripeptide glutathione as well proteins such as β-lactoglobulin and human serum albumin were added to the effluent of the electrochemical cell and allowed to react with the oxidation products. Generated adducts were detected and identified by means of (LC)/HRMS. In case of protein adducts, additional experiments were performed with the aim to obtain further information about the nature and the site of the protein modification. Therefore, the proteins were digested with trypsin and the resulting peptides were analyzed by means of LC/HRMS. Using this approach, the binding of PAH metabolites to free thiol groups in proteins was demonstrated. Neuer Aspekte Reactive PAH metabolites an their protein adducts are generated and characterized by means of EC/LC/MS.
Differential Protein Labeling with Electrochemically Generated Reactive Intermediates Büter, Lars (1,2); Faber, Helene (1); Wigger, Tina (1,2); Karst, Uwe (1,2) 1: Institut für Anorganische und Analytische Chemie, Universität Müsnter; 2: NRW Graduate School of Chemistry, Universität Münster Stichworte: Protein labeling, Mass spectrometry, Electrochemistry, Reactive intermediates, Hyphenated techniques Einleitung In the field of proteomics, the identification as well as the quantification of proteins in complex mixtures is required. The identification of proteins can be performed via the identification of a specific enzymatic peptide. The determination of the number of a certain amino acid in a protein provides additional information, which allows a simplified identification. Therefore, within this study, cysteine residues in proteins were labeled with electrochemically generated reactive intermediates. The formation of reactive quinoid intermediates via dehydrogenation leads to a selective Michael‑type addition of these electrophilic structures to the nucleophilic thiol group of cysteine residues. Electrochemical generation of reactive intermediates of compounds in their native and isotope labeled form provides a defined mass difference of the differentially labeled biomolecules. Experimenteller Teil The hyphenation of an electrochemical cell, equipped with a boron-doped diamond working electrode, to electrospray ionization MS (ESI‑MS) allows the immediate detection of generated peptide adducts. Applying a potential ramp from 0 ‑ 2500 mV and presenting the data in form of three dimensional mass voltammograms enables a broad overview of the generated products. An extension of the instrumental set‑up by means of liquid chromatography (LC) allows the on-line generation, separation and identification of modified proteins. By employing a switching valve for the injection, the EC oxidation and the LC separation can be performed online prior to MS detection. Ergebnisse Electrochemical oxidation of the two phenolic species phenol and 13C6‑phenol or acetaminophen and D4‑ acetaminophen, respectively, resulted in the generation of reactive intermediates. The obtained quinoid structures benzoquinone or N-acetyl-p-benzoquinone imine (NAPQI) were subsequently allowed to react with the tripeptide glutathione (GSH). The formed adducts were identified by means of ESI-MS. With the help of mass voltammograms, the optimum oxidation potential for the generation of reactive intermediates was determined. Furthermore, it could be shown that the native and the isotopic enriched form of the reactive intermediates formed GSH adducts to the same extent. Thus, a defined mass difference for the differentially labeled peptide could be detected in both cases. By adding a protein to the effluent of the EC cell, the online modification of proteins was obtained. In order to separate and identify the protein adducts, EC was coupled to LC/ESI‑MS. The protein carbonic anhydrase I (CAI), which is highly abundant in red blood cells, was incubated with the reactive intermediates. For phenol and 13C6phenol, quantitative conversion of the free thiol groups was achieved. Moreover, only one adduct was formed, which could be traced back to adduct formation of CAI with benzoquinone. In contrast, labeling after oxidation of acetaminophen and D4‑acetaminophen was not quantitative and next to an adduct with NAPQI, a further adduct with a dehydrogenated dimer of acetaminophen was detected. Thus, it could be shown that the system phenol/ 13C6-phenol is better suited for protein labeling than acetaminophen/D4‑acetaminophen. After enzymatic digestion, a mass difference of differentially labeled, cysteine containing peptide was observed This can be traced back to the heavier, labeled species of phenol. Cysteine was identified to be the only binding site in the protein. Based on the obtained mass difference, a simplified identification of cysteine containing peptides in complex mixtures is possible. Neuer Aspekte
Differential labeling and tryptic digestion of proteins allows the detection of a defined mass difference for cysteine containing peptides.
Study on highly aromatic complex mixtures such as crude oil asphaltenes with online and offline SEC-MS Molnárné Guricza, Lilla; Schrader, Wolfgang Max-Planck-Institut für Kohlenforschung Stichworte: Asphaltenes, hyphenated techniques, size exclusion chromatography, high resolution mass spectrometry Einleitung Asphaltenes are the heavy components present in crude oil and exemplify a very diverse fraction. They have a wider range of polarity than other fractions, can be highly aromatic and show different solubilities. Furthermore, asphaltene compounds fairly differ in their molecular sizes.1 Chromatographic separation is an excellent tool to simplify extreme complex mixtures. The sample simplification and reduced matrix effect make possible to get deeper information about samples containing large number of different compounds using mass spectrometry (MS) for detection.2 Size exclusion chromatography (SEC) is the method where the separation is taking place according to hydrodynamic ratio. Therefore, the coupling of SEC and MS offers a good opportunity to effectively analyze asphaltenes on a molecular level.3 Experimenteller Teil Size exclusion chromatography was performed using an Agilent 1100 HPLC system. As optimized separation conditions, a combination of two PSS SDV (styrene-divinyl benzene copolymer) analytical columns (300 x 8.0 mm ID, 5 µm) were used with pore sizes of 1000 Å and 100 Å, respectively as stationary phase and tetrahydrofuran as mobile phase. Mass spectrometric analysis was performed on a research-type LTQ-Orbitrap Elite MS equipped with a combination of APPI and APCI source. In addition to MS, UV detector at 300 nm was used, as well. Ergebnisse For the investigation of highly aromatic complex structures such as asphaltenes, a coupling system of SEC and MS was developed. Compounds present only in small amounts can be detected more sensitively due to the separation. SEC presents the method of choice for separation of asphaltenes because solvents can be used that dissolve the compounds and here the results of online and offline coupling are compared. The separation results only in one but fairly broad chromatographic peak with a time width of 6 min where all components were separated according to their size. The detection of this separation can be done both online - where each second two scans could be recorded - and offline where fractions within a time range of 1 min was collected for detailed analysis. The results show that there still is a remarkable similarity between the online and offline coupling results. The main advantage of offline coupling is that higher mass resolution can be applied, so even deeper information can be obtained, although some separation might be lost due to the fraction collection. The mass range of asphaltene molecules detected by MS is 200-800 Da. The results from the individual fractions cover this whole mass range. Compounds having larger masses are eluting first and the smaller ones are detected later due to the different exclusion from the pores of the packing material. The properties of the compounds from the different fractions during separation are discussed in this presentation. Neuer Aspekte SEC coupling with high resolution MS (online and offline) is applied to analyze highly aromatic complex mixtures such as asphaltenes. Referenzen
[1] O.C. Mullins, E.Y. Sheu, A. Hammami, A.G. Marshall, Asphaltenes, Heavy Oils and Petroleomics. Springer: New York, 2010.; [2] S. Lababidi, S.K. Panda, J.T. Andersson, W. Schrader, Anal. Chem. 2013, 85, 9478-9485.; [3] F. Karaca, C.A. Islas, M. Millan, M. Behrouzi, T.J. Morgan, A.A. Herod, R. Kandiyoti, Energy Fuels 2004, 18, 778-788.
Characterization of a versatile low temperature plasma torch by optical emission spectroscopy and time-offlight mass spectrometry Bierstedt, Andreas; Riedel, Jens BAM, Deutschland Stichworte: Ambient ionization, Low temperature plasma, Dielectric Barrier Discharge (DBD), Emission spectroscopy, ToF-MS Einleitung In recent years, several designs of stable atmospheric pressure plasma sources have been developed and characterized. [1,2] One of the most prevalent devices is the dielectric barrier discharge (DBD), where high voltages in the kHz frequency range are used to ignite a bright plasma between two separated electrodes. The DBD is inexpensive and robust running at low plasma temperatures, resulting in little thermal stress for the sample. The DBD can additionally be used for ambient, solvent-free analysis of untreated samples. Regarding these advantages the DBD is a suitable candidate as a powerful ionization source in mass spectrometry. Our goal is a further understanding of the participating mechanisms and reactions in the plasma by using mass spectrometry and emission spectroscopy. Experimenteller Teil The DBD source consists of a simple toolbox, resulting in the possibility of a rapid change of different working modes. The individual parts are all commercially available mass products and entirely disposable. It involves a borosilicate glass tube, different types of copper mesh as external powered electrode, a stainless steel acupuncture needle as inner grounded electrode and a Swagelok Tee-piece. High voltage is provided by an adjustable input power source and a low cost flyback high voltage transformer. Helium serves as discharge gas at an adjustable flow, controlled by a mass flow meter. All experiments were conducted using an orthogonal time-of-flight mass spectrometer with an atmospheric pressure inlet capillary. Spectral imaging was conducted on an in-house 3D-optical emission spectroscopy tomograph. Ergebnisse The ultimate goal is the use of a plasma source as an effective post-ionization source for mass spectrometry, avoiding at the same time fragmentation and competitive chemistry. For DBDs, several competitive modes of operation resulting in different ionization pathways have been discussed. Our used toolbox allows for a wide variety of simple and easy to change set-ups of the DBD source. In all set-ups, formation of reactive species was found to be highly effective, but further understanding of the participating mechanisms and reactions in the plasma is desired for maximum ionization efficiency with a minimum of subsequent fragmentation. Blank spectra of ambient air show intensive formation of water cluster series, up to higher masses of 1 kDa, as well as smaller contributions of reactive oxygen and nitrogen species. The DBD source was additionally used to desorb and ionize different analyte compounds, e.g. drugs, polymers and organometals, deposited between the exit of the glass tube and the mass spectrometer. Depending on the used setup either the plasma itself or the plasma-activated gas stream exits the glass tube and interacts with the sample directly where it desorbs and ionizes surface molecules in the ambient environment. To yield further insight into the discharge region, a 3D spectral imaging characterization was performed. Spectra were acquired at ~ 70.000 different positions of the plasma, mapping the electronic temperature, electron density and reactive species number densities. A preliminary analysis of the obtained results indicates that the postulated ionization mechanisms need revision. The obtained knowledge of the plasma processes allows us to selectively switch between the formation of only radical cations and only protonated ions. Besides the geometrical arrangement of electrodes, the flow rate seems to have the biggest influence on the formation of the particular species. Neuer Aspekte
Tunable DBD source for the formation of either radical or protonated ions. Spatial and Spectral imaging of DBD source. Referenzen [1] M. Laroussi, T. Akan, Plasma Processes Polym. 2007, 4, 777-788.; [2] C. Meyer, S. Müller, E. L. Gurevich, J. Franzke, Analyst 2011, 136, 2427-2440.
Mass spectrometry of oligopeptides in the presence of alkali halides using desorption/ionization induced by neutral cluster impact Portz, Andre (1); Baur, Markus (2); Heep, Julian (1); Gebhardt, Christoph (3) 1: Justus-Liebig-Universität Giessen, Deutschland; 2: Fakultät Angewandte Naturwissenschaften, Hochschule Esslingen, Deutschland; 3: Bruker Daltonik GmbH, Bremen, Deutschland Stichworte: Soft desorption/ionization, alkali-peptide complexes Einleitung Oligopeptides in natural environment are often exposed to relatively high concentrations of alkali halides, hence the investigation of biomolecules in this particular environment is of high importance. Furthermore, peptides can build non-covalently bonded complexes with metal ions; a promising field of research in this context is the study of metal complexes acting as target-selective proteases catalysing the hydrolytic cleavage of disease-related oligopeptides or proteins [1,2]. In this work we show the applicability of desorption/ionization induced by neutral SO2 clusters (DINeC) for the study of biomolecules in their natural environment as well as for the understanding of their interaction with metal ions. Experimenteller Teil Bare oligopeptides and metal complexes were desorbed and ionized from a SiO2-substrate by neutral SO2 clusters with a mean size of 103 to 104 molecules seeded in a pulsed He beam. The impacting polar clusters both provide the energy for desorption as well as they serve as a transient matrix in which the analyte molecule is dissolved during cluster surface impact. The latter effect lowers the energy barrier for desorption which thus takes place very softly and without fragmentation of the analyte [3,4]. The samples were prepared by drop casting the respective aqueous solution with alkali halide concentrations up to an excess of a factor of 1000 and absolute concentrations up to 1 mol/l. Ergebnisse Using DINeC as desorption/ionization method, clear spectra of the intact biomolecule [M+H]+ as well as of the intact complex of a biomolecule with an alkali ion X, [M+X]+, were detected even in the presence of a large excess of salt in the original solution. The ratio between metal complexes [M+X]+ and bare biomolecules [M+H]+ can be controlled by using different preparation procedures. The effect was traced back to phase separation of alkali halides and biomolecules during the drying cycle of the preparation; the ratio of metal complexes and bare biomolecules was found to decrease within the first main group. Furthermore, DINeC was efficiently combined with ion trap mass spectrometry also allowing for structural analysis of the non-fragmented metal complexes due to its MSn capabilities. The intact metal complexes [M+X]+ and [M+X+H]2+ were isolated and fragmented in the ion trap, the fragments were attributed to respective sequences of Angiotensin II. Both, fragments carrying an alkali ion as well as bare fragments could be identified. Based on the distribution of these fragments, a localization of the metal ion in Angiotensin II molecules was possible. According to our results, alkali ions are mainly bound to the carboxyl groups of aspartic acid and phenylalanine located at the terminal groups of the molecule. The localization and binding strength seems to be stronger for Li and Na compared to K. Neuer Aspekte clear spectra from biomolecule/salt mixtures, desorption of intact complexes of peptides with alkali ions, localization of alkali ion in peptides Referenzen [1] G. Grasso, S. Bonnet, Metallomics 6, 1346 (2014); [2] J. Suh, W.S. Chei, Curr. Opin. Chem. Biol. 12, 207-213 (2008); [3] C. R. Gebhardt, et al., Angew. Chem. Int. Ed. 48, 4162 (2009); [4] M. Baur, et al., Rapid Commun. Mass Spectrom. 28, 290-296 (2014)
Fundamentals and Applications of Plasma-based Ambient Desorption Ionization Mass Spectrometry in Bioanalysis and Lithium-Ion Battery Research Engelhard, Carsten (1); Albert, Anastasia (2); Kuhlmann, Christopher (1); Shelley, Jacob T. (3); Vortmann, Britta (2,4); Nowak, Sascha (2,4) 1: University of Siegen, Germany; 2: University of Muenster, Germany; 3: Kent State University, Kent, USA; 4: MEET Battery Research Center, Muenster, Germany Stichworte: LTP, Orbitrap, ambient desorption/ionization Einleitung Ambient desorption/ionization mass spectrometry (ADI-MS) aims to enable direct analysis of gaseous, liquid, and/or solid samples in the ambient environment. In ADI-MS, different types of desorption/ionization sources are classified according to their basic method of operation, namely spray-based, laser-based, and plasma-based sources. In this presentation, the applicability of plasma-based sources for both direct qualitative and quantitative analysis will be discussed. One benefit of using a desorption/ionization source is the possibility to skip a preceding chromatography run if high mass resolution is available at the detector. This, in turn, significantly reduces the amount of solvents required per analysis and sample throughput improves. Experimenteller Teil A home-built low-temperature plasma probe (LTP, based on the original work by Harper et al. [1]) was coupled to a high-resolution mass spectrometer (HRMS, model Exactive HCD, Thermo Fisher). After careful optimization of source parameters and sample-to-probe geometry [2], desorption/ionization of dried analyte spots is facilitated using the afterglow of the dielectric-barrier discharge that extends out of the LTP probe. Depending on the compounds of interest, additional heating may be applied to aid thermal desorption from the sample target. Ergebnisse Several examples will be discussed, in which LTP-HRMS was found useful for screening applications. In contrast to qualitative analysis, quantitative analysis with plasma-based ADI-MS is still challenging but recent reports indicate that it is very well feasible if methods are developed carefully. Here, a method for rapid and quantitative analysis of pesticides in fruits by QuEChERS pretreatment and LTP-HRMS will be discussed [3]. Lastly, LTP-MS was applied for direct analysis of lithium-ion battery (LIB) compounds. After controlled aging of model electrolytes, degradation products were successfully identified and helped to further understand the reaction mechanisms in LIB [4]. Based on our results, LTP-HRMS is considered a useful and complementary technique for the mass spectrometric toolbox. Neuer Aspekte Quantitative analyses with low-temperature plasma ambient desorption/ionization high resolution mass spectrometry (LTP-HRMS) Referenzen [1] J. D. Harper, N. A. Charipar, C. C. Mulligan, X. Zhang, R. G. Cooks, and Z. Ouyang, Anal. Chem. 2008, 80, 9097-9104; [2] A. Albert and C. Engelhard, Spectrochim. Acta, Part B, 2014, published online, DOI: 10.1016/j.sab.2014.08.034; [3] A. Albert, A. Kramer, S. Scheeren, and C. Engelhard, Anal. Methods, 2014, 6(15), 5463-5471; [4] B. Vortmann, S. Nowak, and C. Engelhard, Anal. Chem. 2013, 85, 3433-3438
LILBID-MS reveals structural insights into a unique hybrid FoVo rotor and its integration into the ATPase Morgner, Nina Uni Frankfurt, Deutschland Stichworte: non-covalent MS, ATPase, C-ring, LILBID Einleitung In recent years mass spectrometry (MS) evolved into a powerful method to analyse the stoichiometry and connectivity of multiprotein complexes. While soluble complexes have long since been targeted successfully by MS, the hydrophobic membrane complexes remained challenging. Especially suited to membrane proteins is the LILBID-MS technique (Laser Induced Liquid bead Ion Desorption-MS). Experimenteller Teil It employs an on-demand droplet generator which introduces 50mm droplets of the analyte into the vacuum where they are irradiated by an IR-laser pulse. This leads to the explosive expansion of the droplet setting the solvated ions free. Then they can be analyzed by time of flight MS. Ergebnisse LILBID allows the measurement even of membrane protein complexes lacking any soluble parts, such as isolated ATPase c-rings. Here I will present results from the Na+ F1FO ATP synthase of A. woodii, which has a unique FOVO hybrid rotor, for which three genes (atpE1, atpE2, atpE3) encode different c-subunits (the V-type like c1 (four trans membrane helices (TMH)) and the identical F-type c2/c3 (two TMH)). Earlier results had shown, that these always result in a c-ring of a fixed stoichiometry of 9:1 (c1 : c2/3). With the help of deletion mutants we could show that subunits c1 and c2 are essential for a functional ATP synthase. MS analysis of the c rings isolated from these ATPase mutants revealed that the c subunit stoichiometry remains unaltered for those mutants that still contain the V-type c1 subunit, while in the case of Dc1 the loss of c1 is compensated for by two additional copies of c2/3. Deletion of an essential c subunit leads to the assembly of incomplete ATPases, which allowed us to investigate the steps involved in the integration of the c ring into the ATPase. Neuer Aspekte analysis of hybrid F0V0 rotor, assembly of c-ring into ATPase
Identification and relative label-free quantification of lactosylated peptides in bovine raw milk and processed milk products Milkovska, Sanja (1,2); Hoffmann, Ralf (1,2) 1: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig; 2: Center for Biotechnology and Biomedicine, Universität Leipzig Stichworte: Amadori product, boronate affinity chromatography, lactosylation, milk processing, relative label-free quantification Einleitung The microbiological safety of milk products is guaranteed by different types of thermal treatment providing also longer shelf life times. These elevated temperatures and the high content of lactose, however, favor Maillard reactions yielding in the early stage Amadori products at lysine residues (lactulosyllysyl). Extensive lactosylation of milk proteins modifies lysine residues and thereby decreases the protein nutritional value and reduces their degradation by enzymes after consumption with heavily glycated sequences being resistant to proteolysis. Furthermore, lactosylated milk proteins may trigger an immune response and thereby trigger food allergies. Although such severe side effects have been discussed, compelling data supporting these concerns are missing demanding comprehensive studies determining the modification sites and degrees of proteins in processed milk products. Experimenteller Teil Lactosylation induced in milk proteins during thermal processing including the modification sites were determined in bovine raw milk (RM), raw colostrum (RC), pasteurized milk (P), ultra high temperature (UHT) milk, and infant formulas (IFs). Proteins were extracted, digested with trypsin, lactosylated peptides were enriched with boronate affinity chromatography, and analyzed by nLC-ESI-MS/MS (CID and ETD mode). A retention time-based inclusion list of all potentially lactosylated peptides was used in a subsequent nLC-ESI-MS/MS (ETD mode) to retrieve the peptide sequences and the modification sites in all milk samples. Lactosylated peptides identified with high confidence scores and additionally manually confirmed were quantified by integration of the corresponding extracted ion current chromatograms (XICs). Ergebnisse In total, we could identify 260 unique lactosylated peptides corresponding 123 lactosylation sites (LS) in 28 proteins. Most peptides were singly lactosylated, but a few di-lactosylated peptides were also identified mostly in UHT and IF. All five analyzed IF samples contained even triply lactosylated peptides indicating higher glycation levels. Correspondingly, the highest number of lactosylated peptides and unique lactosylation sites (106) were identified in IF. Several lactosylation sites were already present in un-processed raw milk. The whey proteins β-lactoglobulin and α-lactoglobulin as well as kappa-casein appeared to contain the most lactosylation sites, although this might be linked to their high concentrations allowing a better analysis. Additionally, all 260 identified lactosylation sites were quantified using the signal intensities of the precursor ions acquired for all milk samples. The quantitative data provided the basis to evaluate the impact of different thermal processes on the glycation degree relative to untreated raw milk. The highest lactosylation degree was determined in infant formulas. UHT contained most lactosylation sites at lower quantities, although the relative content depended very much on the protein. Pasteurized milk contained much lower levels that were similar to RC and RM. Thus, both RC and RM contained already a relatively high basic lactosylation degree that increased only for harsher thermal treatment conditions (UHT) and additional drying (IF). Interestingly, the lactosylation levels varied very much among the three UHT and five IF samples obtained from different brands indicating that the complete technical process and not only the sterilization step determines the overall lactosylation degree in both milk products. Neuer Aspekte Twenty-five novel lactosylation sites were identified in bovine milk proteins including six proteins that were not known to be lactosylated.
Multiplexed 2D MRM-based protein biomarker quantitation of plasma proteins from free flaps – a pilot study Yang, Jing-zhi (1); Percy, Andrew (2); Yang, Jun-Cong (2); Borchers, Christoph (2); Fritschen, Uwe von (3); Finke, Juliane C (3); Glocker, Michael O (1) 1: Proteome Center Rostock, University Medicine and Natural Science Faculty, University of Rostock, Rostock, Germany; 2: University of Victoria - Genome British Columbia Proteomics Center, Vancouver Island Technology Park, Victoria, BC, Canada; 3: Division of Plastic Surgery and Hand Surgery, HELIOS Clinic Emil von Behring, Berlin, Germany Stichworte: Plasma, Protein Quantitation, Multiple reaction monitoring (MRM), Multiplexed, Ischemia and Reperfusion Einleitung Free flap transplantation has become the gold standard to cover complex wounds of the human body. However, socalled “ischemia and reperfusion injuries” of the transplants are unavoidable during surgery; potentially leading to complications and even loss of the transplant. Monitoring tissue-related processes on the molecular level is desired to assess individual transplant quality when clinical signs are not visible [1]. Since blood is circulating all tissues it contains proteins secreted from them. We collected human plasma samples from the feeding artery and the effluent vein of free flaps during surgery to study their respective protein profiles by LC-MRM-MS [2]. In this pilot study we investigated abundances of two proteins (MCSF and calgranulin A) in human plasma samples from eight patients. Experimenteller Teil Proteins in 20µl plasma were denatured, reduced and alkylated. After digesting with trypsin, 5 stable-isotope-labelled standard (SIS) peptides were spiked-in. Peptide mixtures were desalted by solid phase extraction, then eluted with 600 µl 55% ACN in 0.1% FA and lyophilized. For injection onto the high-pH RPLC system, peptides were rehydrated with 1600 µl 10mM ammonium hydroxide. After LC fractionation of the peptide mixture into 47 wells of a 96 well plate, eluates were lyophilized. Before analysis at low-pH RPLC/MRM-MS, neighboring fractions were combined into 13 fractions for each sample in 100 µl of 0.1% FA. Fractions were analyzed on a tripe quadrupole mass spectrometer (6490; Agilent technologies), MRM data were processed with Mass Hunter Quantitative Analysis software (version B. 07.00). Ergebnisse Five proteotypic peptides were chosen to represent MCSF and calgranulin A. Chromatographic peaks were manually inspected for peak-related information (retention time, peak width and area) to ensure correct selection of MRM fragments for integration. For quantitation the peak area filter was set to 300 counts. Peaks areas above 300 were determined to calculate protein concentrations. For MCSF, the peptide FNSVPLTDTGHER with [M+3H]3+ ion signal (m/z 491.58) and its y7+ fragment ion (m/z 815.37), was chosen. The corresponding SIS peptide ([M+3H]3+ at m/z 494.91) and its y7+ fragment (m/z 825.37) was used as reference. Retention time of these peptides is 5.7 min at low pH, and the optimal collision energy is 17 V. For calgranulin A, the selected peptide was GADVWFK ([M+2H] 2+ at m/z 411.71, SIS peptide: m/z 415.72) and its y3+ ion was recorded at m/z 480.26 (SIS fragment: 488.27). Retention time of these peptides is 8.6 min, and the optimal collision energy is 9 V. Using these two transitions it was possible to determine precise quantities of both proteins in all 16 plasma samples. The averaged MCSF concentration in artery plasma was 21.2 ng/mL (± 6.4), whereas that in venous plasma was 32.0 ng/mL (± 9.3); a 1.5-fold upregulation (p < 0.05). Calgranulin A concentration in artery plasma was 3.4 ng/mL (± 1.3), whereas that in venous plasma was 12.8 ng/mL (± 10.9); a 3.75-fold upregulation (p < 0.05). Standard deviations reflect biological variances. MCSF has been reported to increase during tissue inflammation, indicating macrophage activation in the flaps [3]. Calgranulin A, as part of calprotectin, has been discovered to control macrophage activity during
ischemia/reperfusion, causing fibrosis and tissue damage [4]. Our study proves that the risk of tissue damage during transplantation can be monitored by LC-MRM-MS and has the potential for early assessment of free flap quality. Neuer Aspekte MRM-MS determines differential expression of MSCF and calgranulin A from free-flaps during transplantation for early tissue quality assessment. Referenzen [1] J.M. Smit, C.J. Zeebregts, et al. Advancements in free flap monitoring in the last decade: a critical review, Plast Reconstr Surg, 125 (2010) 177-185; [2] A.J. Percy, R. Simon, et al. Enhanced sensitivity and multiplexing with 2D LC/MRM-MS and labeled standards for deeper and more comprehensive protein quantitation, J Proteomics, 106 (2014) 113-124; [3] Y.L. Meur, G.H, Tesch, et al. Macrophage accumulation at a site of renal inflammation is dependent on the MCSF/c-fms pathway, J. Leukoc. Biol, 72 (2002) 530-537; [4] M.C Dessing, A. Tammaro, et al. The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion, Kidney International, 87 (2015) 85 – 94.
Rapid extraction of intact proteins from tissues via desorption by impulsive excitation Kwiatkowski, Marcel (1); Wurlitzer, Marcus (1); Omidi, Maryam (1); Nimer, Refat (1); Kruber, Sebastian (2); Miller, R.J. Dwayne (2); Schlüter, Hartmut (1) 1: Mass Spectrometric Proteomics, Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf (UKE), Germany; 2: Atomically Resolved Dynamics Department ,Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany Stichworte: protein extraction, tissue samples, PIRL-DIVE, proteomic, mass spectrometry Einleitung Protein extraction from tissue is a critical and time-consuming step in every protein analysis. Proteins must be released from their native environments using homogenization and further isolation methods, which vary depending on the nature of the tissue and proteins of interest. An appropriate protein extraction procedure shouldn't change their chemical compositions and functions like enzymatic activities. Proteolytical and other degradations should be avoided during the extraction process. In this work we examined the potential of a picosecond-IR laser (PIRL) [1] to extract proteins from tissue samples through desorption by impulsive excitation (DIVE) of intramolecular vibrational states of water molecules [2]. Experimenteller Teil To test the suitability of DIVE for protein extraction, several experiments were performed. First, mouse muscle and liver tissue samples were irradiated by PIRL. The condensates of the ablation plumes were analyzed by SDS-PAGE to investigate whether a broad range of proteins can be extracted by PIRL-DIVE and if these are suitable for immunoblotting. Furthermore, the integrity and enzymatic activity of ablated proteins were investigated by mass spectrometry. For this purpose PIRL-desorbeded and non-desorbed RNase A were analyzed on peptide and protein level to investigate the chemical compositions of the ablated molecules. The enzymatic activities of PIRL desorbed trypsin and human blood plasma were investigated by mass spectrometry after incubation with α-casein respectively angiotensin I (Ang 1-10). Ergebnisse With DIVE almost 1 mg of proteins were extracted from 37.5 mm3 mouse muscle tissue in less than five minutes, whereas the conventional extraction procedure lasted more than three days with a total protein yield of 175 µg. The SDS-PAGE of the PIRL plume condensate showed a huge number of bands covering proteins from a few kDa up to several hundred kDa. LC-MS/MS analysis of selected bands revealed that even large proteins like titin (approx. 4 MDa) were extracted in a huge quantity. In addition, PIRL protein extracts were generated from mouse liver tissue for immunoblotting. Detection of highly glycosylated CEACAM1 protein showed that even labile PTMs survived the DIVE process. A detailed investigation of the chemical composition of RNase A in the PIRL plume condensate by MALDI-MS and nanoUPLC-ESI-QTOF-MS/MS on peptide and protein level revealed that the chemical composition wasn't altered by DIVE. Furthermore, enzymatic activities of DIVE treated trypsin as well as DIVE treated human blood plasma proteins were retained. LC-MS analysis of the incubate of trypsin with α-casein showed a number of signals for tryptic α-casein peptides, whereas no signals for the intact α-casein protein were observed. Detection of Ang 1-8, Ang 6-9, Ang 4-10 and Ang 1-7 in the incubate of the condensate of the DIVE plume of human blood plasma with Ang 1-10 showed that Ang 1-10 metabolizing enzymes were still active after PIRL irradiation. Since MALDI spectra of the incubate of ablated blood plasma and non-ablated blood plasma with Ang 1-10 showed very similar patterns, it can be assumed that almost all of the enzyme activities were maintained after DIVE extraction. In summary, DIVE facilitates an ultrafast protein extraction from tissues in a high quantity and quality. DIVE is compatible with common protein analytical techniques, minimizes time and increases the yield of extracted proteins. Neuer Aspekte PIRL-DIVE is a new, ultrafast and innovative method for extracting proteins from tissues in a high quantity and quality.
Referenzen [1] Franjic K, Cowan ML, Kraemer D, Miller RJD. Laser selective cutting of biological tissues by impulsive heat deposition through ultrafast vibrational excitations. Opt Express, 2009, 17, 22937-59.; [2] Kwiatkowski M, Wurlitzer M, Omidi M, Ren L, Kruber S, Nimer N, Robertson WD, Horst A, Miller RJD, Schlüter H. Ultrafast Extraction of Proteins from Tissues Using Desorption by Impulsive Vibrational Excitation. Angew. Chem. Int. Ed., 2015, 54, 285-88.
Flowsystem on-line gekoppelt mit einem LC/qTOF-MS zur systematischen Untersuchung der Photolyse unter umweltrelevanten Bedingungen Seiwert, Bettina; Weidauer, Cindy; Reemtsma, Thorsten Helmholtz-Zentrum für Umweltforschung, Deutschland Stichworte: Transformationsprodukte, Pathway, Photolyse, Flowsystem, Spektren-Datenbank Einleitung Transformationsprodukte (TP) in komplexen Umweltproben zu detektieren und einzelnen Chemikalien zuzuordnen ist aufgrund der Vielzahl möglicher Reaktionen und Produkte nicht einfach. Noch schwieriger wird es, wenn man auch Aussagen über Pathways machen möchte. Daher ist es sinnvoll, einzelne Transformationsprozesse im Labor nachzustellen und schrittweise deren Komplexität zu erhöhen. Für den photolytischen Abbau von Carbamazepin gibt es in der Literatur unterschiedliche Pathways [1, 2, 3]. Diese wurden vor allem basierend auf den teilweise nicht vollständig bestätigten Strukturvorschlägen der Abbauprodukte aufgestellt. Das Fließsystem ist in der Lage schnell eine systematische Untersuchung von Ausgangsstoffen und seinen Transformationsprodukten vorzunehmen. Eine gemeinsame Analyse der erhaltenen LC/MS-Spektren erleichtert dann das Aufstellen von Pathways und kann über Plausibilitätsprüfungen und gemeinsamer Analyse der MS/MS-Spektren auch zu einer verbesserten Strukturaufklärung beitragen. Experimenteller Teil Das Flowsystem besteht aus einer Anordnung von Spritzenpumpen und Ventilen, die über eine gemeinsame Software gesteuert werden. Grundlage ist ein kontinuierlicher Fluss, in den die Analyte (eventuell mit bestimmten Zusätzen) als Plugs injiziert werden . Diese Plugs werden dann in einer Schnecke aus FEP-Schlauch eingeleitet und von einer Xenonlampe, die sich in einer Kammer befindet, die thermostatiert werden kann, gleichzeitig bestahlt. Über die Flussrate lässt sich die Bestrahlungsdauer verändern. Des Weiteren kann auch die Bestrahlungsstärke der Xenonlampe variiert werden. Über einen Loop erfolgt die Injektion der einzelnen Plugs in das LC/MS-System, wo diese mit einer schnellen, kurzen Gradienten mittels UPLC/HRMS vermessen werden. Die Auswertung der LC/MSChromatogramme der Analyte und deren Analoga findet dann gemeinsam mit Unifi (Waters) statt. Ergebnisse Die online-Kopplung des Photolyse-Systems zu einem LC-qTOF ermöglicht ein systematisches schnelles Screening von anthropogenen Spurenstoffen und deren Transformationsprodukten, wodurch man auch für vermeintlich gut untersuchte Substanzen wichtige Zusatzinformationen erhält. Dies soll nun im Folgenden am Beispiel von Carbamazepin (CBZ) und dessen Analoga verdeutlicht werden. Durch Vergleich der LC/MS-Chromatogrammen mit und ohne Bestrahlung kann man eine Aussage zur generellen Abbaubarkeit der Ausgangssubstanzen unter umweltrelevanten Bedingungen vornehmen. CBZ-epoxid und auch das dihydro-diOH-CBZ werden durch direkte Photolyse innerhalb von 3h nicht abgebaut, während Oxcarbazepin zu 100% abgebaut wird. Dies beweist das CBZepoxid nicht ein zentrales TP im Pathway darstellen kann und unter umweltrelevanten Bedingungen nicht zur Bildung von Acridin und BQD führt, wie teilweise in der Literatur [1] vermutet wurde. Die Auswertung der LC/MSChromatogramme findet für CBZ und dessen Analoga (Ox-CBZ, 3-OH-CBZ, diOH-CBZ, Epoxid-CBZ, Acridin) gleichzeitig statt, wodurch die Identifizierung bekannter und neuer unbekannter TPs wesentlich erleichtert wird. Diese Strategie verdeutlicht auch, dass gleiche exakte Massen (m/z 224.0706) und damit gleiche Summenformeln (C14H9NO2) aus den Analoga entstehen, die aufgrund ihrer Retentionszeit aber unterschieden werden können. Eine Zuordnung eines Strukturvorschlages allein aufgrund der Fragmente ist teilweise nur mit dem Wissen über die Ausgangstoffe möglich. Ein Ziel des Flowsystems ist es dabei auch eine Datenbank von Transformationsprodukten zu erstellen, in der Informationen wie Summenformeln, Retentionszeit, Isotopenmuster und MS/MS- Spektren gespeichert sind. Diese Informationen können dann für ein umfassendes Umweltmonitoring genutzt werden. Die zusätzliche Information über den oder die Ausgangsstoffe, die zum jeweiligen TP führen, kann dabei wertvolle Hinweise geben. Neuer Aspekte
Flowsystem zur on-line-Generierung von TPs analoger Substanzen, die zusammen ausgewertet werden erleichtert die Strukturaufklärung und ermöglicht ein verifizieren aufgestellter Pathways. Referenzen [1] E. De Laurentiis et al., Environ Sci Technol 2012, 46 (15), 8164-8173; [2] S. Chiron et al., Environ Sci Technol. 2006 Oct 1;40(19):5977-83; [3] E. Calza et al., Environ Sci Pollut Res Int. 2013 Apr;20(4):2262-70
On-line Analysis of Organic Emissions from Residential Wood Combustion with Single-Photon Ionization Time-of-Flight Mass Spectrometry (SPI-TOFMS) Czech, Hendryk (1); Sippula, Olli (2,3); Kortelainen, Miika (2); Tissari, Jarkko (2); Streibel, Thorsten (1,3); Zimmermann, Ralf (1,3) 1: Joint Mass Spectrometry Centre University of Rostock, Chair of Analytical Chemistry, Germany and HelmholtzZentrum München, CMA (Comprehensive Molecular Analytics), Germany; 2: University of Eastern Finland, Department of Environmental Science, Fine Particle and Aerosol Technology; 3: Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health (HICE) - www.hice-vi.eu Stichworte: single-photon ionization (SPI), non-negative matrix factorization (NMF), principal component analysis (PCA), wood combustion, burning phase Einleitung According to Directive 2009/28/EC, the Commission of the European Union fixed targets for its member states to reach a mean percentage of 20% renewable energy of the final energy consumption in the whole European Union until 2020, for example by the combustion of wood. Therefore wood combustion, in particular during winter, contributes significantly to indoor and outdoor air pollution. Wood smoke and wood combustion-derived particles are known for their toxicological effects. Thus, it is necessary to characterise the emission of different wood combustion appliances, whose characteristics change qualitatively as well as quantitatively with ongoing advances on wood stoves. However, time-resolved measurements for volatile and intermediate-volatile organic compounds (VOC and IVOC) are limited. Experimenteller Teil Six consecutive batches of three common European types of firewood (beech, birch and spruce; batch weight: 2.5 kg; total duration: 4 h) were burned in a modern wood log fired masonry heater. The temporal variation of emissions of single organic compounds was studied by single-photon ionization time-of-flight mass spectrometry (SPI-TOFMS) with 118 nm VUV-photons. SPI refers to a soft ionization technique, thus leading to molecular ions, low fragmentation and no adducts [1]. Every compound with ionization energy below the photon energy becomes ionized whereby the ionization efficiency strongly depends on the compound class [2]. VUV-photons of 118 nm were generated by multiple frequency increasing of the fundamental radiation of 1,064 nm of a Nd:YAG-laser (energy of 40mJ @355nm). Ergebnisse Emissions of the whole combustion cycle and temporal variations were discussed. Short-chain carbonyls, alkenes and dienes, furan/isoprene and benzene as nonspecific decomposition products revealed highest abundances, but m/z of phenolic species and anhydrous sugars as building blocks of the wood polymers lignin, cellulose, and hemicellulose were also observed. Regarding the different batches, more than 50% of the overall intensity for all considered m/z occurred during the combustion of the first two batches. Mainly m/z of components with not less than one double bond and low O/C ratio were significantly enhanced in the first or first two batches, which was ascertained by single and double Grubb’s Outlier Test. Furthermore, the high temporal resolution down to 1s facilitates to investigate the combustion process more in detail. Within one batch, two different temporal trends were perceived: The first group of m/z diminished steadily, whereas the second group had its maximum at the beginning as well, but passed through a minimum and increased again towards the end of the batch. Mainly m/z of benzene, naphthalene, butadiene, vinylacetylene, and propene belong to the second group, remaining m/z to the first group. In one spruce experiment, generally increased intensities were observed, but not defrayed in equal proportions of all m/z. Compared to another spruce experiment, primary decomposition products of wood polymers are more enhanced than secondary decomposition products, such as benzene or small alkenes. Finally, the molecular signatures of burning phases (‘ignition’, ‘stable combustion’ and ‘ember’) were examined by using non-negative matrix factorization (NMF) [3,4] and principal component analysis (PCA) in sequence. Marker substances for wood or biomass combustion, such as phenolic species or anhydrous sugars, exhibited highest relative abundance during ‘stable combustion’, whereas ‘ember’ was distinctly characterized by unsaturated hydrocarbons, such as benzene or naphthalene, through pyrosynthesis; in ‘ignition’, secondary decomposition products dominated.
Neuer Aspekte Burning phases of wood were determined by NMF from VOC and IVOC and subsequently characterized on a molecular level. Referenzen [1] L. Hanley and R. Zimmermann, Analytical Chemistry 81, 4174 (2009).; [2] T. Adam and R. Zimmermann, Analytical and Bioanalytical Chemistry 389, 1941 (2007).; [3] M. W. Berry et al., Computational Statistics and Data Analysis 52, 155 (2007).; [4] M. Elsasser et al., Energy and Fuels 27, 4959 (2013).
Automated fast screening of TOF-MS data Weggler, Benedikt (1,2,4); Jennerwein, Maximilian (2,3); Gröger, Thomas (1,2); Zimmermann, Ralf (1,2,4) 1: 1aJoint Mass Spectrometry Centre, Cooperation Group “Comprehensive Molecular Analytics” Helmholtz Zentrum Muenchen,; 2: GermanybJoint Mass Spectrometry Centre, Institute of Chemistry, Chair of Analytical Chemistry University of Rostock; 3: ASG Analytik Service GmbH, Trentiner Ring 30, 86356 Neusäss; 4: Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health – Aerosol and Health (HICE) Stichworte: Automated Compound Classification, Fast Screening, Quantification Einleitung Sophisticated separation techniques like GCxGC-TOFMS are well established for the comprehensive characterization of complex sample compositions that contain a large number of compounds. Due to the complex amount of information the data evaluation is difficult and time consuming. To cope with this, advanced scripting algorithms based on knowledge based rules(KBR) were in house developed and applied to GCxGC TOFMS data. Thereby, thousand different compounds can reliable be classified in a short time period based on their fragmentation pattern. The quantification of this classes is challenging. Based on the response factors and group specific response curves of a set of clearly separable standards and carbon numbers the ultra-fast screening method could be acomplished by a comprehensive quantification of these compounds. Experimenteller Teil For the analysis by GCxGC-TOFMS, an Agilent 6890gas chromatograph equipped with a LECO Pegasus 4D detector,including a Pegasus IV TOF-MS (LECO, USA) using helium as the carrier. An Optic 3 inlet system(ATAS GL, Netherlands) was used. Data collection was performed using LECO ChromaTOF® software v. 4.50.8. Chromatographic separation was performed on a 60 m × 0.25-mm i.d. × 0.25- µm df BPX5(SGE, Australia) column and then a 1.9 m × 0.1-mm i.d. × 0.10- µm df BPX50 (SGE, Australia) column Ergebnisse If implemented properly, this automated screening approach allows samples withover 4000 peaks to be screened in less than 20 min without adeep knowledge of the sample composition. Compared to previously published approaches the accuracy of the classification algorithms could beincreased significantly by adding fragmentation patterns, molecular ion information and specific molecular fragments. Combining this approach with an interpolation of response factors or an average response, covering the main compounds and carbon numbers is possible and can be used for a nearly complete quantifycation of e.g. middle distillates. Neuer Aspekte Automated Compound classification abased on fragmentation pattern Group-type quantification
Separation of Carbohydrate and Glycoprotein Isomers using Ion Mobility-Mass Spectrometry Hofmann, Johanna (1,2); Hinneburg, Hannes (2,3); Hahm, Heung S. (2,3); Kolarich, Daniel (2,3); Seeberger, Peter H. (2,3); Pagel, Kevin (1,2) 1: Fritz Haber Institute of the Max Planck Society, Berlin, Deutschland; 2: Institute for Chemistry and Biochemistry, Free University Berlin, Berlin, Germany; 3: Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Potsdam, Germany Stichworte: Ion mobility-mass spectrometry, glycans, protein glycosylation, collision cross section, travelling wave Einleitung Complex carbohydrates are of great importance for a variety of biological functions. They can be linked to proteins as post-translational modifications or exist as free oligosaccharides, for example in milk. Currently, the vast majority of complex carbohydrates are characterized using mass spectrometry-based techniques (MS) that are directly descending form those established in proteomics. Measuring the molecular weight of a sugar, however, immediately poses a fundamental problem: entire classes of the constituting monosaccharide building blocks exhibit an identical atomic composition and, consequently, also an identical mass.[1] Therefore, carbohydrate MS data can be highly ambiguous and often it is simply not possible to clearly assign a particular molecular structure. Experimenteller Teil A promising approach to overcome the above-mentioned limitation is to implement an additional gas phase separation step using ion mobility-mass spectrometry (IM‑MS). Here, ions travel through a gas-filled cell aided by an electric field and are separated according to their mass, charge, and shape. While the drift time of an ion depends on the underlying instrument conditions, the collision cross section (CCS) is a molecular property that can be compared and calculated theoretically. Due to the inhomogeneous travelling wave field utilized in most of the commercially available instruments, however, CCS estimation requires careful calibration.[2,3] Ergebnisse Here, we demonstrate the potential of IM-MS to be used as a tool for the separation and identification of isomeric carbohydrates and glycopeptides. First, six synthetic carbohydrate isomers that differ with respect to their composition, connectivity, or configuration were analyzed individually and as mixtures. Our data reveal that carbohydrate linkage- and stereoisomers, which are difficult to distinguish using established techniques such as liquid chromatography or NMR, can be separated and unambiguously identified on basis of their CCS. When mixed, even minor isomeric components with relative concentrations as low as 0.1% were still clearly detectable. Second, we recently extended our investigations to glycopeptides. First preliminary data show that the position of the carbohydrate within the peptide sequence can be clearly identified using IM-MS when multiple glycosylation sites are present. More importantly, however, our data reveal that also glycopeptides, which merely differ in the regiochemistry of the attached glycan can be distinguished using gas-phase fragmentation and subsequent IM-MS analysis. These results indicate that IM-MS is an exceptionally effective tool for the structural analysis of complex carbohydrates. The full benefit of this method will become apparent once CCS data of carbohydrates and oligosaccharide fragments will be deposited in databases. Neuer Aspekte Analysis of carbohydrate isomers using ion mobility-mass spectrometry. Referenzen [1] K. Mariño, J. Bones, J. J. Kattla, P. M. Rudd, Nat. Chem. Biol. 2010, 6, 713-723.; [2] K. Pagel, D. J. Harvey, Anal. Chem. 2013, 85, 5138-5145.; [3] J. Hofmann, K. Pagel et al. Anal. Chem. 2014, 86, 10789-10795.
Influence of glycoforms on the tryptic digestion efficiency of immunoglobulin G based biopharmaceuticals Falck, David (1); Plomp, Rosina (1); Jansen, Bas J. (1); Reusch, Dietmar (2); Haberger, Markus (2); Wuhrer, Manfred (1,3) 1: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; 2: Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany; 3: Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands Stichworte: Bottom-up proteomics, Glycoform profiling, Biopharmaceuticals, nanoLC–MS, Tryptic digestion biases Einleitung Bottom-up proteomics is employed frequently in biological and clinical research. Nowadays, it is becoming increasingly popular for the analysis of biopharmaceuticals in discovery, development and batch control. The digestion of the target protein by proteases, generally by trypsin, into more easy to analyse peptides is a key step in bottom-up proteomics. Thus, a robust analysis needs to incorporate an efficient and unbiased tryptic digestion step. It has been observed previously that incomplete tryptic digestion can introduce biases into glycoform profiles[1]. Therefore, we investigated the preferential tryptic digestion of glycoforms of two biopharmaceutical formats: 1.an immunoglobulin G1(IgG1) monoclonal antibody(mAb) and 2.Flebogamma, an intravenous IgG(IVIG) from healthy donor pools. Via denaturation experiments, we collected evidence for the 3D-structure dependence of the biases. Experimenteller Teil The tryptic digestion of the target protein into glycopeptides was followed over time. The digestion of protein with an intact 3D-structure was compared to that of protein denatured by various methods. These included denaturation, reduction and alkylation as a gold-standard as well as a novel, save and efficient protocol for IgG denaturation. Glycopeptides from the conserved glycosylation site in the FC-part of IgG were detected by a nanoLC–ESI-q-TOF-MS method[2]. In addition, the glycoproteins and glycopeptides of partially digested samples were separated by gel electrophoresis and the residual glycoproteins were reanalysed after complete in-gel tryptic digestion. By relative quantitation of the glycopeptides and estimation of their absolute abundances, we could visualize a preference of trypsin for the digestion of certain glycoforms. Ergebnisse In general, we proved that the glycoforms of the IgG1 mAb containing high mannose and hybrid type glycans in the Fc portion were preferentially digested into the glycopeptides compared to those glycoforms containing complex type glycans. The biases observed in IVIG were smaller, because this formulation did not contain high mannose or hybrid glycans. The main bias in IVIG was created by preferential digestion of bisected species. Interestingly, the α2-3 linked sialic acid containing species in the IgG1 mAb were amongst the first to be digested while the α2-6 linked sialic acid containing species in IVIG were not preferred. Comparing the tryptic digestions of non-denatured and denatured protein revealed that the biases are much less pronounced in denatured samples. Additional experiments proved that this effect is independent of the increased digestion efficiency. A significant part of the digestion biases must therefore be caused by the differences in antibody 3D structure induced by the different glycoforms. Our simple, novel pre-treatment method — acid incubation and evaporation — exhibited a denaturation efficiency comparable to the gold standard (see Methods) for IgG while significantly reducing the hands-on time and the toxicity of the reagents involved. It additionally offers the advantage of performing the tryptic digestion under optimal buffer conditions. In conclusion, proper denaturation and digestion completeness are key to avoiding biases in bottom-up proteomics based glycoform profiling. However, the digestion biases have implications beyond glycoproteomics. For example, relative protein quantitation (even with isotopically labelled standards) in two samples could be biased by differences in glycoform profiles, if complete tryptic digestion is not achieved. Neuer Aspekte
Trypsin preferentially digests certain glycoforms of IgG. This preference is extensively induced by the specific 3D structure of the glycoform. Referenzen [1] [1] Du et al. (2012), Journal of Chromatography B, 907:87-93; [2] [2] Selman et al. (2012), Journal of Proteomics 75(4):1318-1329
MALDI TOF and ESI IT MS Characterization of highly sulfated Glycosaminoglycan-Oligosaccharides Lemmnitzer, Katharina (1); Blaskiewicz, Joanna (2); Schiller, Jürgen (1); Rademann, Jörg (2) 1: Institut für Medizinische Physik und Biophysik, Medizinische Fakultät, Universität Leipzig; 2: Institut für Pharmazie, Freie Universität Berlin Stichworte: sulfated glycosaminoglycans, oligosaccharides, loss of sulfation Einleitung Glycosaminoglycans (GAG) are natural, acidic polysaccharides which occur in the extracellular matrix of many tissues [1]. Certain growth factors and cytokines are known to bind to GAG. This process is assumed to be of significant relevance for wound healing and tissue regeneration [2]. However, the role of the sulfation pattern regarding GAG-protein interactions is unknown. Thus, well characterized GAG (oligosaccharides) with defined sulfation patterns are urgently required for further studies. The mass spectrometric characterization of these highly sulfated compounds is challenging due to the lability of the sulfate residues. Here, the characteristics of a persulfated (ps) tetrasaccharide of hyaluronan (HA-4) will be investigated by ESI IT and MALDI TOF MS and compared with the model compound Arixtra. Experimenteller Teil Arixtra (as 12 mg/mL solution in physiological saline) was purchased from Glaxo Smith Kline and either directly used as supplied or after exhaustive dialysis to reduce the salt content. The persulfated HA-tetrasaccharide (psHA-4) was (obtained by digestion of the HA polysaccharide exhaustive sulfation with sulfur trioxide pyridine complex.
obtained from by hyaluronidase)
HA-4 by
MALDI TOF mass spectra were acquired on a Bruker Autoflex MS and ESI IT spectra on a Bruker Amazon SL. 9Aminoacridine (9-AA) is the matrix of choice to record negative ion spectra due to its pK of about 10. Selected positive ion spectra were also recorded in the presence of DHB. For ESI MS a mixture of methanol and distilled water (2:1, v/v) was used as solvent system. Ergebnisse ESI and MALDI are "soft" ionization techniques and can be widely used in structural studies. (Highly) sulfated carbohydrates are very refractive and normally result in poor ion yields and/or loss of sulfate residues [3]. To verify the capabilities and limitations of both MS methods to characterize newly synthesized sulfated carbohydrates, a reliable reference compound is unequivocally needed. "Fondaparinux" (trade name "Arixtra"), a synthetic, commercially available pentasaccharide with 8 sulfate residues, is a very useful compound for that purpose. Arixtra could be detected by MALDI MS in negative and positive ion mode without the need of desalting, i.e. in the presence of physiological salt concentration. We could show that the negative ion mode provides generally higher sensitivity whereas the intact ion is detectable as positive ion with higher intensity. Additionally, the number of sodium counter ions determines the number of detectable sulfate residues, i.e. the MALDI spectral quality decreases with a decreasing salt concentration. The inevitable [4], significant loss of sulfate during the MALDI process complicates the evaluation of newly synthesized compounds. Since ESI MS is a "softer" method it is more suitable to determine the extent of sulfation [5] although the presence of salt suppresses the ionization process. After exhaustive dialysis, Arixtra could be detected as intact ion with 8 (53%) sulfate residues although loss of one (33%) and more than one (14%) sulfate residue does also occur. Similar data can be obtained when the persulfated HA-4 is investigated. However, the intensity of the intact persulfated Hyaluronan-Tetrasaccharide ion accounts only for 11%. We are currently working on the establishment of an improved desalting method to exclude partial sulfate loss during the dialysis process.
Neuer Aspekte This is the first (MALDI and ESI) mass spectrometric study of such artificially highly sulfated oligosaccharides. Referenzen [1] Schiller J, Huster D.: New methods to study the composition and structure of the extracellular matrix in natural and bioengineered tissues. Biomatter, 2 (2012) 115-131.; [2] Schnabelrauch M, Scharnweber D, Schiller J.: Sulfated glycosaminoglycans as promising artificial extracellular matrix components to improve the regeneration of tissues. Curr. Med. Chem., 20 (2013) 2501-2523.; [3] Kovensky J.: Sulfated oligosaccharides: new targets for drug development? Curr. Med. Chem., 16 (2009) 23382344.; [4] Laremore TN, Zhang F, Linhardt RJ.: Ionic liquid matrix for direct UV-MALDI-TOF-MS analysis of dermatan sulfate and chondroitin sulfate oligosaccharides. Anal. Chem., 79 (2007) 1604-1610.; [5] Jen CH, Leary JA.: A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry. Anal. Biochem., 407 (2010) 134-140.
Combining thin-layer chromatography, antibody-overlay detection and DESI-FT-ICR-MS for the analysis of Shiga toxin glycosphingolipid receptors Steckhan, Katja (1); Große Kracht, Christoph (1); Veith, Lothar (2); Engelhard, Carsten (3); Steil, Daniel (1); Meisen, Iris (1); Müthing, Johannes (1); Mormann, Michael (1) 1: Institute for Hygiene, University of Münster, Robert-Koch-Straße 41, 48149 Münster, Germany; 2: Tascon GmbH, Mendelstraße 17, 48149 Münster, Germany; 3: Institute for Analytical Chemistry, University of Siegen, AdolfReichwein-Straße 2, 57068 Siegen, Germany Stichworte: DESI, FT-ICR, glycosphingolipids Einleitung Glycosphingolipids (GSLs) are integral part of the cell membrane of vertebrates and play important roles in many biological processes [1]. They participate in cell recognition and serve as receptors for numerous pathogens [2] or virulence factors, such as Shiga toxins (Stxs) released by enterohemorragic E.coli (EHEC) [3]. GSLs are comprised of a polar oligosaccharide head group as well as an unpolar ceramide part made of a fatty acid and sphingosine. Structural characterisation of the molecular heterogeneity demands a combination of separation techniques such as thin-layer chromatography (TLC) and mass spectrometry. Here, we demonstrate the use of desorption electrospray ionisation [4] Fourier-transform ion cyclotron resonance mass spectrometry (DESI-FT-ICR-MS) for the analysis of GSLs separated by TLC and detected by immunostaining techniques. Experimenteller Teil GSLs from human erythrocytes were applied bandwise onto normal phase silica TLC plates, separated, and detected by antibody-overlay assays. To that end plates were incubated with either a specific anti-GSL antibody or a Stx, its respective toxin-specific antibody followed by incubation with a secondary antibody and staining with 5-bromo-4chloro-3-indolyl phosphate (BCIP) giving rise to bright blue bands. For DESI measurements, the plate was placed onto a movable sample stage and analytes were desorbed and ionised directly from the plate by use of methanol as a solvent spray. Mass analysis was done with a Fourier-transform ion cyclotron resonance mass spectrometer equipped with a 7 T magnet. A CO2 laser was used for infrared multiphoton dissociation (IRMPD) MS2 experiments. Ergebnisse GSLs could be separated according to their oligosaccharide chain lengths, e.g. globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer). Furthermore, separation depending on the length and saturation of the fatty acid was possible to some extent. GSLs were detected using anti-GSL antibodies, alkaline phosphatase (AP) conjugated secondary antibodies and the staining reagent BCIP. Alternatively, GSL receptors could be detected specifically by Stxs and the latter were recognised by AP conjugated secondary antibodies. Here we show the use of Stx2a which binds preferentially to Gb3Cer. Stained bands were examined by DESI-FT-ICR-MS. Analytes could be desorbed and ionised directly from the TLC plate and were detected even in the presence of other biomolecules such as antibodies and staining reagent. Scanning the plate in the direction of chromatographic development furnished 2D spectra which showed the distribution of the different GSL species on the plate. Scanning of just a single band gave rise to mass spectra of unseparated GSLs of similar polarity. Due to the high mass accuracy of the FT-ICR mass spectrometer all ionic species could be assigned to GSL structures. Gb4Cer detected by antibody-overlay assays could be detected with good signal-to-noise-ratios at amounts down to 20 ng per band. For further structure elucidation, precursor ions were isolated in the ICR cell and fragmented by IRMPD. MS/MS experiments yielded B- and Y-type ions from cleavages of the glycosidic bonds, as well as typical fragment ions allowing characterisation of the sphingosine portion. The combination of TLC and DESI-FT-ICR-MS offers a suitable alternative to conventional methods for GSL analysis such as ESI-MS or MALDI-MS. DESI provides desorption and ionisation of analytes without preceding sample preparation, thus, analyte loss is largely reduced. Ions can be probed very accurately with the FT-ICR mass spectrometer and MS/MS experiments enable structure assignments. Neuer Aspekte
TLC and antibody-overlay detection in combination with DESI-FT-ICR-MS is a new, alternative method for the analysis of glycosphingolipids. Referenzen [1] I. Meisen, M. Mormann, J. Müthing, Biochim. Biophys. Acta 2011, 1811, 875–896.; [2] A. Müsken, J. Souady, K. Dreisewerd, W. Zhang, U. Distler, J. Peter-Katalinić, H. Miller-Podraza, H. Karch, J. Müthing, Rapid Commun. Mass Spectrom. 2010, 24, 1032–1038.; [3] J. Müthing, I. Meisen, W. Zhang, M. Bielaszewska, M. Mormann, R. Bauerfeind, M. A. Schmidt, A. W. Friedrich, H. Karch, Glycobiology 2012, 22, 849–862.; [4] Z. Takáts, J. M. Wiseman, R. G. Cooks, J. Mass Spectrom. 2005, 40, 1261–1275.
Massenspektrometrische Analyse von Biotensiden am Beispiel der Rhamnolipide Behrens, Beate (1); Engelen, Jeannine (2); Hayen, Heiko (1) 1: Westfälische Wilhelms-Universität Münster, Deutschland; 2: Bergische Universität Wuppertal, Deutschland Stichworte: Biotenside, Rhamnolipide, LC-MS/MS, GC-MS, SPE Einleitung Rhamnolipide (RL) gehören zu den Tensiden mikrobiellen Ursprungs. Sie zeichnen sich durch eine geringe Toxizität und eine gute Bioabbaubarkeit aus und eignen sich daher z.B. für die Anwendung als Emulgatoren in Kosmetika und Lebensmitteln. RL bestehen aus ein bis zwei Rhamnoseeinheiten (mono-/di-RL), welche mit bis zu drei βHydroxyfettsäuren mit einer Kettenlänge von C8-C14 verbunden sind. Die mikrobielle Produktion der RL in Gemischen sowie ihre strukturelle Vielfalt erfordern effiziente analytische Methoden, um qualitative und quantitative Informationen über die Zusammensetzung der RL-Probe zu erhalten. Chromatographische Methoden gekoppelt mit einer massenspektrometrischen Detektion haben sich für die Identifizierung von RL als geeignet erwiesen [1,2]. Experimenteller Teil Für die Analyse der Rhamnolipid-Proben wurde die HPLC gekoppelt mit der Tandem-Massenspektrometrie (LCMS/MS) angewendet. Die freie Carboxylgruppe ermöglicht die Ionisierung mittels Electrospray (ESI) im negativen Modus. Für die Charakterisierung der RL-Proben wurden verschiedene Scan-Experimente (Full Scan, ProduktionenScan, Vorläuferionen-Scan und MRM) herangezogen. Zusätzlich wurde die GaschromatographieMassenspektrometrie (GC-MS) mit Elektronenionisation zur Bestätigung der identifizierten Rhamnolipide anhand der Zusammensetzung ihrer β-Hydroxyfettsäuren eingesetzt. Ergebnisse Die analysierten Rhamnolipide wurden durch den Mikroorganismus Pseudomonas putida KT2440 in einem Fermentationsprozess hergestellt [3]. Das zellfreie Probenmaterial wurde durch die Arbeitsgruppe von Prof. Blank (RWTH Aachen) zur Verfügung gestellt. Als Alternative zur bisher üblichen Flüssig-Flüssig-Extraktion wurde die Festphasenextraktion (SPE) als Probenvorbereitung angewendet. Die SPE wurde hinsichtlich der Durchführung der Wasch- und Elutionsschritte optimiert. Die quantitative Elution aller Analyten ist von der Konzentration der Rhamnolipide im Probenmaterial abhängig und wird durch die Mischung Acetonitril/Ameisensäure (≥ 2%) erreicht. Nach der Aufarbeitung erfolgte die Analyse mittels HPLC-(ESI-)MS und HPLC-(ESI-)MS/MS. Mono-RL verschiedener Kettenlängen wurden anhand ihrer deprotonierten Molekülionen [M-H]- und ihrer spezifischen Fragmente in MS/MS-Experimenten identifiziert und in ihrer relativen Verteilung bestimmt. Zusätzlich wurde die komplementäre Analyse der Fettsäuren mittels GC-MS nach Derivatisierung zu den korrespondierenden Fettsäuremethylestern durchgeführt. Die Identifizierung erfolgte über die Retentionszeiten von Standards und spezifischen Fragmenten aus der Elektronenionisation. Mit den genannten Methoden wurde ein Rhamnolipid mit der seltenen Zusammensetzung von Rhamnose (Rha) verbunden mit β-Hydroxydecansäure (C10) und βHydroxyundecansäure (C11), Rha-C10-C11 und das Isomer Rha-C11-C10, identifiziert. Die Existenz eines Rhamnolipids mit dieser Zusammensetzung wurde bisher in der Literatur nur einmal erwähnt und konnte nicht mit anderen Methoden als der HPLC-MS bestätigt werden [4]. Neuer Aspekte Anwendung der Festphasenextraktion als Alternative zur bisher üblichen Probenaufarbeitung und Identifizierung eines seltenen Rhamnolipids mittels komplementärer LC-MS/MS- und GC-MS-Experimente Referenzen [1] Heyd, M., Kohnert, A., Tan, TH., Nusser, M., Kirschhöfer, F., Brenner-Weiss, G., Franzreb, M., Berensmeier, S.; Anal. Bioanal. Chem., 391 (2008) 1579-1590.; [2] Zgoła-Grześkowiak, A., Kaczorek, E.; Talanta, 83 (2011) 744-750.;
[3] Wittgens, A., Tiso, T., Arndt, TT., Wenk, P., Hemmerich, J., Müller, C., Wichmann, R., Küpper, B., Zwick, M., Wilhelm, S., Hausmann, R., Syldatk, C., Rosenau, F., Blank, LM.; Microb. Cell Fact., 10 (2011) 80.; [4] Noordman, WH., Brusseau, ML., Janssen, DB.; Environ. Sci. Technol., 34 (2000) 832-838.
Bestimmung von polaren Metaboliten in Algenextrakten mittels HILIC-ESI-MS Schriewer, Alexander; Hayen, Heiko Westfälische Wilhelms-Universität Münster, Deutschland Stichworte: HILIC-ESI-MS, Redox-Cofaktoren, Flussanalyse, Probenvorbereitung von Algenzellen Einleitung Cofaktoren wie NAD+/NADH, NADP+/NADPH oder FAD/FADH2 sind essentiell für die Redox-Homöostase. Des Weiteren spielen Metabolite wie Glutathion, ATP, ADP und AMP eine wichtige Rolle im Zellgleichgewicht. Glutathion kann freie Radikale abfangen und so die Redox-Fähigkeit der Zellzyklen zu regulieren,[1] während ATP der universelle und essentielle Energieträger in jeder Zelle ist. Zur Bestimmung der Redoxzustände und der Regenerationsrate von Cofaktoren in Zellen und Organismen ist es notwendig, deren Flussverteilung in Stoffwechselwegen zu bestimmen. Das Ziel der Flussanalyse ist die Quantifizierung von metabolischen Flüssen im Zentralstoffwechsel von Mikroorganismen.[2] Die Ermittlung der Flussverteilung kann durch 13C- und/oder 15N-Markierungsexperimente im Zellwachstum und anschließender massenspektrometrischer Analyse der Verhältnisse unterschiedlich markierter Analyten erfolgen. Experimenteller Teil Bei der Methodenentwicklung wurde der Fokus auf die Metabolite FAD, NAD+, NADH, NADP+, NADPH, AMP, ADP, ATP sowie Glutathion in oxidierter und reduzierter Form gelegt. Die Hydrophile Interaktionsflüssigkeitschromatographie (HILIC) bietet eine Möglichkeit polare bis sehr polare Analyten chromatographisch zu separieren. In Verbindung mit einem hochauflösenden Massenspektrometer können zudem verschieden isotopenmarkierte Analyten unterschieden werden. Die Entwicklung einer Trennung wurde mit Standards auf einer HILIC-Säule durchgeführt, während die Detektion im negativen ESI-Modus erfolgte. Die Anwendbarkeit der entwickelten Methode wurde anhand von Algenextrakten getestet, die aus Chlamydomonas reinhardtii-Kulturen nach Zellaufschluss und anschließender Flüssig-Flüssig-Extraktion gewonnen wurden. Zum Aufschluss der Zellen wurden verschiedene Verfahren evaluiert, die die Zellwände mechanisch zerstören, um die intrazellulären Metabolite für die Extraktion zugänglich zu machen. Ergebnisse Mithilfe der HILIC konnte eine Trennung der zumeist sehr polaren Cofaktoren erfolgreich entwickelt werden. Bei der Entwicklung der Trennung wurden neben verschiedenen HILIC‑Säulen auch Mixed-Mode‑Säulen getestet. Dazu wurden sowohl die Zusammensetzung der mobilen Phase als auch die Pufferkonzentration und der pH-Wert des Puffers verändert. Zur optimalen Trennung wurde der Anteil des organischen Lösungsmittels sowie die Pufferkonzentration in der Probenlösung variiert. Bei dieser Untersuchung konnten die besten Ergebnisse mit einem Acetonitril-Anteil von 75% und einem 50 mM Ammoniumacetat-Puffer (pH 5,76) erreicht werden. Die Modellkultur Chlamydomonas reinhardtii, bereitgestellt von der Arbeitsgruppe von Prof. Michael Hippler (Westfälische Wilhelms-Universität Münster, Institut für Biologie und Biotechnologie der Pflanzen), wurde dazu verwendet verschiedene Aufarbeitungsmethoden miteinander zu vergleichen. Zum einen wurde ein Zellaufschluss nach Wase et al. mit Methanol, Chloroform und Wasser durchgeführt.[3] Zum anderen wurde ein Aufschluss mittels Kugelmühle und anschließender Flüssig-Flüssig-Extraktion mit Wasser und Chloroform angewendet.[4] Weiterhin wurde der Aufschluss mit einem Nebulizer sowie durch Ultraschall durchgeführt. Das Potential der vorgestellten Methode zur 13C- bzw. 15N-basierten Flussanalyse wird am Beispiel der Bestimmung der Markierungsgrade von Metaboliten in Algenextrakten demonstriert. Neuer Aspekte
HILIC-ESI-MS Trennung von zehn polaren Redox-Cofaktoren in einem Lauf zur Flussanalyse mittels LC-ESI-MS. Referenzen [1] A. Pompella, A. Visvikis, A. Paolicchi, V. D. Tata, A. F. Casini, Biochem. Pharmacol. 2003, 66, 1499-1503; [2] W. Wiechert, Metab. Eng. 2001, 3, 195-206; [3] N. Wase, P. N. Black, B. A. Stanley, C. C. DiRusso, J. Proteome Res. 2014, 13, 1373-1693; [4] D. Y. Lee, O. Fiehn, Plant Methods 2008, 4, 7
Bringing Triple Quadrupole Detection Limits to a New Standard –in Theory and in Practice Falter, Ralf; Glauner, Thomas; Toelgyesi, Laszlo Agilent Technologie, Deutschland Stichworte: S/N, IDL, detection limits, instrument detection limits Einleitung
We report on the use of an instrument detection limit (IDL) based on random standard deviation (RSD) of a series of replicates as a universal measure of “ion efficiency” and analytical utility. The IDL methodology for LC/MS is illustrated for the analysis of samples from food safety and environmental applications and data show a significant improvement in limits of quantitation. The obtained IDL levels are therefore independent of the noise measurement as long as the noise comes as a small fraction of the measured signal. Experimenteller Teil Sample preparation Extracts of black tea obtained using QuEChERS and dispersive SPE were diluted 1:20 with acetonitrile and then spiked with several pesticides presenting high risk relevance in the concentration range of 0.2ppt to100ppb. Steroids spiked in neat solvents were directly injected into the mass spectrometer using an injection volume of 20μL. Chromatography was performed under gradient conditions using 2.1x100mm columns-Eclipse-Plus C18 (1.8μm particle size) and Poroshell120 (2.7μm particle size) for pesticide and steroid analysis, respectively. Ergebnisse More specifically, due to the random nature of the ion flow to the detector the standard deviation (SD) of the number of arrivals in a unit of time follows that of the Poisson distribution, and depends on the average number of ions N as Neuer Aspekte S/N definition compared to Instrument Detection Limit (IDL) methode
Identification of urinary peptides triggering social behavior of Danio Rerio by mass spectrometry Markgraf, Astrid (1); Christoffers, Jens (2); Gerlach, Gabriele (1) 1: Institute of Biology and Environmental Sciences, Carl von Ossietzky University, D- 26111 Oldenburg, Germany; 2: Institute of Chemistry and Center of Interface Science, Carl von Ossietzky University, D-26111 Oldenburg, Germany Stichworte: Peptides, zebrafish, MHC, olfactory signals, chemical signals Einleitung Imprinting in zebrafish is a learning process that is limited to a short time period during early development and leads to an irreversible change in behavior [1]. In a narrow time window (day 6 post fertilization) zebrafish larvae imprint on olfactory cues of siblings, which results in a preference of kin over non kin [2]. The biochemical mechanisms underlying this complex process are poorly understood. Particularly, the triggers which are necessary to impress a lifelong memory remain elusive. However, accumulating data suggests that major histocompatibility complex (MHC) dependent peptides, released by the urine, are implicated in olfactory imprinting. Experimenteller Teil We kept different group of siblings (30 zebrafishes) in 1.5 liters freshwater for 24 hours. Subsequently we performed a solid-phase extraction to enrich the peptide concentration and to separate them from electrolytes and low-molecular matrices. This extract was probed with an ESI-Q-TOF mass spectrometer to get distinct primary peptide sequences out of the diverse mixture of molecules. Afterwards the zebrafish groups were genotyped for their MHC class II alleles by SSCP gel to find secreted peptide specific for the respective MHC background. Ergebnisse Proteins of the major histocompatibility complex (MHC) play a fundamental role in discriminating ‘self’ and ‘nonself’ in the immune system by presenting pathogen-derived short peptides to lymphocytes. In vivo calcium imaging shows, that some olfactory bulb neurons are highly tuned to MHC peptides with a detection threshold at 1 pM or lower, demonstrating that fish can smell MHC peptides at physiological concentration [3].The aim of this project is to identify putative signal peptides in the urine of zebrafish involved in olfactory imprinting. In comparison with several zebrafish families with different MHC background, we want to limit the identified set of urinary peptide to single peptides specific for the respective MHC type. Neuer Aspekte For the first time we were able to identify peptides in the urine of zebrafish presumably involved in olfactory imprinting. Referenzen [1] Gerlach G, Hodgins-Davis A, Avolio C, Schunter C (2008) Kin recognition in zebrafish: A 24-hour window for olfactory imprinting. Proceedings of the Royal Society of London B 275 2165–2170.; [2] Hinz C, Müller A, Kobbenbring S, Sigman L, Gerlach G (2013a) Kin recognition in zebrafish, Danio rerio, is based on imprinting on olfactory and visual stimuli. Animal Behaviour 85, 925-930.; [3] Hinz C, Namekawa I, Behrmann-Godel J, et al. (2013b) Olfactory imprinting is triggered by MHC peptide ligands. Scientific Reports 3 : 2800.
Advancements in (MA)LDI based mass spectrometry imaging techniques in the field of chemical ecology Kaftan, Filip (1); Weißflog, Jerrit (1); Pande, Samay (2); Kulkarni, Purva (3); Napagoda, Mayuri (4); Jaschinski, Tino (5); Svatoš, Aleš (1) 1: Max Planck Institute for Chemical Ecology, Deutschland; 2: Institute of Integrative Biology, Department of Environmental Systems, Zürich, Switzerland; 3: Chair for Bioinformatics, Friedrich Schiller University Jena, Jena, Germany; 4: Faculty of Medicine, University of Ruhuna, Department of Biochemistry, Sri Lanka; 5: Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Germany Stichworte: MALDI MSI, AP-SMALDI MSI, Drosophila melanogaster, surface lipids, cVA, CH5O3, 1, 8Di(piperidinyl)-naphthalene, 14-Diaza[5]helicene Einleitung The field of chemical ecology investigates the various ways in which organisms interact chemically. For example symbioses, parasitism, chemical defense agents, attractions of pollinators etc. Imaging techniques are important tools providing information about the exact location of production, storage and release of these chemicals. Especially techniques based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) are suited for the analysis of wide spectrum of biological samples (e.g. plants, insects, microorganisms) giving high spatial resolution and detailed information about the chemical compounds present in the tissues, cells or other biological structures. Experimenteller Teil Despite of the advancement of these MALDI based techniques the low amount of present compound and the 3-D character of the sample require an improvement in sensitivity and prevention of mass shift 1 because of the non-planar character of the sample. Additionally we are focused on small molecules, some of which are not easily ionizable. Therefore we were designing and screening for novel matrix systems to improve sensitivity and expand the scope of possible analytes. Furthermore we are employing state of the art of hardware e.g. AP-SMALDI Orbitrap based MS to push the limits in terms of spatial and mass resolution, giving new insights in several ecologically relevant issues. Ergebnisse 1) We found that the simply pre-purified commercially available diatomaceous earth2 (Celite) applied by spraying, mediates the laser desorption ionization (LDI) of surface lipids on Drosophila melanogaster flies. Using this approach, very clean mass spectra were obtained without any interfering signals which are related to Celite. 2) Two novel matrices, 1,8-Di(piperidinyl)-naphthalene and 1,14-Diaza[5]helicene3, showed improved sensitivity especially towards lipid based analytes. Furthermore these matrices showed high vacuum stability making them useful for prolonged MALDI MSI experiments in a negative mode. Owing to their superbasic nature they are able to deprotonate weak acidic analytes in solution already during sample preparation. Thus the ionization takes place prior to the laser desorption. The matrices themselves are not deprotonated, which prevents the formation of interfering signals. 3) MALDI MSI technique was used to investigate cross-feeding interactions in bacteria biofilms via spatial distribution of amino acids (histidine and tryptophan) which were formed into crystals on the surface of the biofilm. A synthetic system was constructed between Acinetobacter baylyi and Escherichia coli, in which both strains reciprocally exchange amino acids. The MALDI MSI results were supported by HR MS analysis of surface crystals. The spatial distribution of cocultured cross-feeding cooperators was investigated by fluorescence microscopy. Neuer Aspekte Commercially available diatomaceous earth Celite was succesfully used for ionization of embedden molecules in LDI MSI experiments of D. melanogaster. Referenzen [1] Kaftan F. et al. J. Mass Spectrom. 2014, 49, 223-232.;
[2] Jaschinski T. et al. Rapid Commun. Mass Spectrom. 2013, 27, 109–116; [3] Napagoda, M. et al. ChemPlusChem 2013, 78, 937-942.
Determination of the platinum distribution in Caenorhabditis elegans after Cisplatin incubation by LA-ICPMS Crone, Barbara (1); Bornhorst, Julia (2); Aschner, Michael (3); Schwerdtle, Tanja (2); Karst, Uwe (1) 1: Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany; 2: Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany; 3: Department of Molecular Pharmacology, Neuroscience, and Pediatrics, Albert Einstein College of Medicine, Bronx NY, USA Stichworte: Bioimaging, LA-ICP-MS, C. elegans, Cisplatin Einleitung Cisplatin is one of the most important and frequently used cytostatic drugs within the treatment of cancer. The cytostatic effect is based on binding to the DNA in tumor cells by forming intra- and interstrand crosslinks. The associated DNA deformations and the interference with DNA repair mechanisms lead to apoptosis in cancer cells. Poly(ADP-ribose)polymerase-1 (PARP-1) is a protein that is involved in several biological pathways including DNA repair mechanisms. The nematode Caenorhabditis elegans (C. elegans) is occupying the poly(ADPribose)metabolism enzyme 1 (pme-1), which is an ortholog of human PARP-1. Loss of this enzyme is resulting in a disturbed DNA damage response. The bioavailability of Cisplatin in C. elegans was analyzed by laser ablationinductively coupled plasma-mass spectrometry (LA-ICP-MS). Experimenteller Teil Experiments were performed using a laser ablation system based on a Nd:YAG laser with a wavelength of 213 nm, coupled to a quadrupole-based inductively coupled plasma mass spectrometer. Besides the dry aerosol, a ruthenium solution as the internal standard was simultaneously introduced, to improve the plasma stability. Possible interferences of e.g. [40Ar155Gd] were minimized in the kinetic energy discrimination mode (KED) with a He/H2 gas mixture as cell gas. The ablation of the nematodes was performed in a multiline scan. Laser parameters were optimized to obtain optimal spatial resolution and elemental information. Furthermore, to validate these data a total reflection x-ray fluorescence (TXRF) method was applied. Ergebnisse The distribution of platinum in C. elegans after Cisplatin incubation was determined. Therefore, L4 stage wildtype worm and pme-1 deletion mutants were treated with different concentrations of Cisplatin. Characteristics of this animal, such as its genetic manipulability and the well-characterized genome makes it a suitable model system. The uptake of Cisplatin was investigated by visualization of the distribution of platinum. Thus, images with a lateral resolution of 5 µm were generated. The elemental mapping indicated that Cisplatin is located in the head of the worms. Furthermore, it was shown that higher Cisplatin concentrations and longer incubation times led to higher platinum concentrations within the worm. Neuer Aspekte The uptake and accumulation of Cisplatin in C. elegans were analyzed successfully by LA-ICP-MS.
Exploring the head and neck cancer by MALDI FT-ICR mass spectrometry imaging Krasny, Lukas (1); Hoffmann, Franziska (2); Ernst, Günther (2); Kobarg, Jan Hendrik (3); Trede, Dennis (3,4); Alexandrov, Theodore (4,5,6); Havlicek, Vladimir (1); Guntinas-Lichius, Orlando (7); von Eggeling, Ferdinand (2,7,8); Crecelius, Anna C. (9,10) 1: Institute of Microbiology, v.v.i., Prague, Czech Republic; 2: Institute of Physical Chemistry, Friedrich Schiller University Jena, Jena, Germany; 3: Steinbeis Innovation Center SCiLS Research, Bremen, Germany; 4: SCiLS GmbH, Bremen, Germany; 5: Center for Industrial Mathematics, University of Bremen, Bremen, Germany; 6: European Molecular Biology Laboratory, Heidelberg, Germany; 7: Department of Otorhinolaryngology, Jena University Hospital, Jena, Germany; 8: Leibniz Institute of Photonic Technology (IPHT), Jena, Germany; 9: Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Jena, Germany; 10: Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Jena, Germany Stichworte: MALDI, MSI, FT-ICR, Head and neck, Cancer Einleitung Head and neck cancer comprises of malignant tumors located in the oral cavity, the throat, and the upper aerodigestive tract. To improve individualized therapy, the spatial molecular characterization is crucial. The aim of this study is to use MALDI mass spectrometry imaging (MALDI imaging) to discover the spatial lipidome of head and neck cancer. Experimenteller Teil MALDI imaging enables the simultaneous analysis of hundreds of molecular compounds in a single measurement. In this study, head and neck cancer samples have been analyzed with a Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer in the mass range of m/z 200 to 1,500 and a spatial resolution of 50 μm. Consecutive sections were stained by H&E and co-registered with the MALDI imaging datasets. For computational evaluation the spatial segmentation approach was used which provides spatial features by statistically determine similarities between spectra. The resulted segmentation map was overlaid with the histological image and validated by an experienced pathologist. A subsequent comparative analysis yielded discriminating m/z values. Ergebnisse We performed the MALDI FT-ICR imaging workflow together with the computational evaluation on four different cancer samples. The high mass resolution of the FT-ICR imaging datasets yielded large data sets, each of a size of about 100 GB. The clusters of the spatial segmentation coincide with morphological structures apparent in the microscopy images. Subsequently, co-localized masses have been found, which discriminate prominent morphological structures. The results show that the morphological changes observed in the investigated head and neck tumor samples are predominantly reflected in the MALDI FT-ICR imaging data. We found m/z values that discriminate different morphological areas. The high mass accuracy and high mass resolving power of FT-ICR mass spectrometer enables to match the discovered peaks against lipidomics databases. Neuer Aspekte The spatial segmentation approach applied for lipidomic analysis reveals new insights into the metabolic organization of carcinoma tissue. Referenzen [1] Alexandrov T, Becker M, Deininger SO, Ernst G, Wehder L, Grasmair M, von Eggeling F, Thiele H, Maass P: J. Proteome Res., 2010, 9:6535-6546; [2] Alexandrov T, Becker M, Guntinas-Lichius O, Ernst G, von Eggeling F: J. Cancer Res. Clin. Oncol., 2013, 139:85-95;
[3] Krasny L, Hoffmann F, Ernst G, Trede D, Alexandrov T, Havlicek V, Guntinas-Lichius O, von Eggeling F, Crecelius AC: J. Am. Soc. Mass Spectrom., 2015, 26:36-43
Laser ablation dielectric barrier discharge imaging MS for direct molecular analysis of pharmaceuticals in tablets Elseberg, Tim (1); Herdering, Christina (1); Sperling, Michael (2); Karst, Uwe (1) 1: University of Münster, Institute of Inorganic and Analytical Chemistry; 2: European Virtual Institute for Speciation Analysis (EVISA) Stichworte: Laser ablation, molecular imaging, Pharmaceuticals, Dielectric barrier discharge, small molecules Einleitung Worldwide 10% of all used drugs are counterfeits.[1] To identify counterfeits, several parameters, e.g., the total amount and lateral distribution of the active pharmaceutical ingredient (API), appearance, packing or excipients can be compared with the original product. While most of these parameters can be analyzed by several analytical methods, the analysis of the surface distribution of the API is generally limited to spectroscopic methods and MALDI-MS.[2] Laser ablation (LA) coupled to a dielectric barrier discharge interface (DBDI) and orbitrap MS is used to obtain high resolution molecular images. The information of these imaging experiments can be used to identify counterfeits via their API distribution and gather further information of the ingredients homogeneity. Experimenteller Teil Two commercially available pain killers with the API caffeine (50 mg), acetylsalicylic acid (250 mg) and acetaminophen (200 mg) were used as test substances. These tablets were placed inside the ablation cell without any sample pretreatment. A 213 nm Nd:YAG laser with a spot size of 25 µm is used to linewise ablate material from a surface. This material is transported with a nitrogen gas stream to a dielectric barrier discharge where it is ionized. The ions are detected by a high resolution orbitrap mass spectrometer. Extracted ion chromatograms of the API are aligned with a housemade software to generate high resolved molecular images in mass and space. Ergebnisse The commonly used methods to analyze the lateral API distribution comprise some disadvantages. LA-DBDI-MS is able to overcome these limitations. The high resolution mass spectrometer offers a new dimension of sample information in contrast to the spectroscopic methods. The exact m/z value allows the identification of an API by its accurate mass. To gather this information, contrary to MALDI experiments, no sample pretreatment, suchs as matrix application, is needed. Neuralgin® and Thomapyrin® are analyzed as test substances by LA-DBDI-MS. The two respective active pharmaceutical ingredients are caffeine, acetylic salicylic acid and acetaminophen with identical concentration, although different excipients are used. It is investigated whether this is reflected in the surface distribution of the API. Therefore, molecular images of caffeine, acetylsalicylic acid and acetaminophen are recorded for both, Neuralgin® and Thomapyrin® and compared regarding their surface homogeneity. It was shown that LA-DBDI-MS is a suitable tool for molecular imaging and that this method is promising to identify counterfeit drugs by deviations in the lateral distribution of the API compared to the original product. Furthermore, this method could help to optimize manufacturing routes to increase the homogeneity of the active components and thereby improve the intake into the human body and the stability of the tablet. Neuer Aspekte High resolved molecular MS imaging of small molecules in pharmaceutical preparations by LA-DBDI-MS without sample pretreatment. Referenzen [1] Wertheimer, A. I.; Norris, J. Res. Social Adm. Pharm. 2009, 5, 4–16.;
[2] Earnshaw, C. J.; Carolan, V. A.; Richards, D. S.; Clench, M. R. Rapid Commun. Mass Spectrom. 2010, 24(11), 1665-1672
Improved spatial resolution in the analysis of FFPE tissue after tryptic digestion Beckmann, Janine; Suckau, Detlev; Becker, Michael; Asperger, Arndt Bruker Daltonik GmbH, Bremen, Deutschland Stichworte: imaging, FFPE, MALDI, brain tissue Einleitung In recent years, MALDI imaging has proven to be a powerful method for clinical research. Although imaging of protein distributions has had substantial impact in this area, three challenges does still have to be addressed: the detection of high molecular weight (>30kDa), the identification of m/z signals and the accessibility of formalin-fixed, paraffin embedded (FFPE) tissues. Trypsin digestion is seen as a promising approach for improving the above points and we present here an update on our efforts to develop a robust protocol for on-tissue digestion. Experimenteller Teil Mouse and rat brains tissue sections were mounted on conductive glass slides. Digestion was preceded by paraffin removal and antigen-retrieval treatment. Enyzme and matrix application was performed by a piezoelectric sprayer. Incubation at elevated temperatures (37 – 50 °C) was conducted in a custom-made humid chamber. MS data was acquired on a MALDI-TOF instrument operating in reflector mode. Results were visualized and evaluated using commercially available software. Ergebnisse On-tissue digestion adds more complexity to sample preparation for MALDI Imaging, which makes development of a robust protocol challenging. Spatial resolution, number of peptides generated and identification rate are considered important for a successful experiment, but optimizing these can be diametrically opposed. We found that high humidity and extended digestion time increased the effectiveness of the digestion, but also on-tissue delocalization of peptides. Short (90%) and reproducible ( 800.000 @ 400 Th zur massenspektrometrischen Analyse von Erdölen unter Nutzung der "Spectral Stitching Methode".[1] Experimenteller Teil Zur Durchführung der Studie wurde ein schweres Erdöl zunächst in Toluol auf 250 ppm verdünnt. Die erhaltene Lösung wurde anschließend per Direktinjektion nach Photoionisation bei 10,0/10,6 eV an einem Orbitrap Elite Prototypen jeweils wie folgt vermessen: - Full Scan im Massenbereich 200 - 1200 Th - Spectral Stitching im Bereich 200 - 1200 Th; Segmentbreite 30 Th; Überlapp 5 Th Für drei verschiedene Transienten/Auflösungen (768 ms; 1,5 s; 3 s) wurden dabei jeweils insgesamt 250 Einzelscans aufgenommen und aufsummiert. Die Interpretation der Daten erfolgte mit Hilfe von Composer (Sierra Analytics, Version 1.0.6). Ergebnisse Für die massenspektrometrische Analyse von Erdölen sind auf instrumenteller Seite zwei Faktoren von entscheidender Bedeutung: Empfindlichkeit bzw. Ionentransmission und eine hinreichend hohe Auflösung über einen weiten Massenbereich. Der klassische Ansatz mittels FT-ICR-Massenspektrometrie verspricht, eine entsprechende Magnetfeldstärke vorausgesetzt, hier ausgezeichnete Resultate. Nachteilig wirken sich jedoch die verminderte Ionentransmission, insbesondere in niedrigen Massenbereichen, und die mit steigendem m/z reziprok abfallende Massenauflösung aus. Orbitrap Elite Massenspektrometer erlauben ab Werk eine maximale Transientendauer von 768 ms, was einer Auflösung von 240k @ 400 Th (FWHM) entspricht. Im von uns verwendeten, darauf basierenden, Prototypen kann die Transientendauer darüberhinaus verdoppelt, bzw. auf ca. 3 s vervierfacht werden. Dies erlaubt unter realen Bedingungen eine Auflösung von >800k @ 400 Th (nominell 960k). Diese Auflösung liegt nur noch geringfügig unter der von FT-ICR-Geräten, die bei einer Feldstärke von 7T die Marke von 1.000.000 erreichen. Durch den gegenüber der FT-ICR-Technik schwächer ausgeprägten Auflösungsverlust bei steigendem Massenbereich wird bei 1.000 Th noch immer eine Auflösung von ca. 500k erreicht. Von besonderem Interesse im Bereich der Erdölanalytik ist die massenspektrometrische Unterscheidung zwischen C3 und SH4, was einer Massendifferenz von 3,4 mDa entspricht. Eine Auflösung von 500k im Bereich von 1.000 Th erlaubt es, diese Unterscheidung noch bis zu einem Peakintensitätsverhältnis von etwa 1:10 sicher zu treffen. Durch die Anwendung des "Spectral Stitching" kann, bei gleichbleibender Gesamtmessdauer, dia Anzahl beobachteter und zugeordneter Signale um den Faktor 3-4 erhöht werden. Der im Vergleich zu einer analogen Studie mit einem FT-
ICR-Massenspektrometer geringe Zugewinn kann der grundsätzlich besseren Ionentransmission zugeschrieben werden.[1] Der Informationsgewinn ist bei allen untersuchten Auflösungen ähnlich. Bei längeren Transienten steigt die Gesamtzahl der zugeordneten Signale erwartungsgemäß hauptsächlich im Bereich über 600 Th aufgrund der zugewonnen Massenauflösung. Neuer Aspekte FT-Orbitrap-MS kann durch erhöhte Auflösungen als günstige und schnelle Alternative zur FT-ICR-MS für Untersuchungen komplexer Proben verwendet werden. Referenzen [1] A. Gaspar, W. Schrader, Rapid Commun. Mass Spectrom. 2012, 26, 1047–1052
Assessing the Peptide Quantitation Performance of a Newly Developed Triple Quadrupole Instrument Falter, Ralf; Gnau, Volker; Miller, Christine Agilent Technologie, Deutschland Stichworte: QQQ, Peptide Quanfication, Proteine Digests Einleitung Sample complexity and low concentration of some biomarkers are the main challenges in biomarker verification methodology based on multiple reaction monitoring (MRM). Consequently, the development of MRM-based methods with stable isotope-labeled standards peptides in biological fluids has focused on improving method sensitivity and increasing the dynamic range by lowering detection limits. Furthermore robustness and reproducibility are essential for using these assays in high-throughput translational research environments. This work demonstrates the sensitive and robust determination of human plasma peptides using a newly designed triple quadrupole mass spectrometer. Improvements include a new optimized Q1 ion transfer optics and a novel ion detector that uses a high voltage conversion dynode with low noise characteristics. Experimenteller Teil
Improved Signal with Higher HED Voltage
•The impact of the high voltage dynode on response was measured using HED voltages of -10, -15, -18 and -20 kV •Results (average of n=3, shown as % gain over the -10 kV) to the right demonstrate a clear benefit to the higher HED voltage (30-80% increase in response at -20 kV) Anti-HER2/neu mAb Digest Following the same protocol as for the enolase experiment, a mAb digest was analyzed in order to test higher m/z product ions and glycopeptide behavior. •Results (average of n=3, shown as % gain over the -10 kV) to the right demonstrate a clear benefit to higher HED voltage (60-120% increase in response at -20 kV) for higher m/z ions
Ergebnisse •The 6495 QQQ with Agilent JetStream Technology provides outstanding sensitivity at standard flow rates with an LLOQ of 5 amol on-column •Ultimate sensitivity is achieved with nanoflow LC/MS on the HPLC-Chip/6495 where we achieved a 500 zmol LLOQ •With a complex plasma digest matrix, the 6495 QQQ showed excellent response and retention time reproducibility over and extended period (0.5 mL of plasma digest injected over 3.5 weeks!) •Improved performance in peptide quantitation can be achieved by the increased precursor ion transmission, enhanced detection efficiency (HED voltage up to -20kV) and extended m/z range of the 6495 QQQ Neuer Aspekte Peptide Quantification with high accuracy
Eine DBD zur in-situ Erzeugung von Reaktantionen für negative chemische Ionisation Albrecht, Sascha (1); Stroh, Fred (1); Benter, Thorsten (2) 1: Forschungszentrum Jülich GmbH, Deutschland; 2: Bergische Universität Wuppertal, Deutschland Stichworte: Dieleektrisch behindertes Plasma, Ionenquelle, Chemische Ionisation Einleitung Die chemische Ionisation (CI) bei Atmosphärendruck wird zumeist noch mit radioaktiven Ionenquellen betrieben, da diese einen hohen und konstanten Ionenstrom liefern. CI-Massenspektrometer werden unter anderem für die Messung atmosphärischer Spurengase verwendet. Da diese Geräte mobil sein müssen sind radioaktive Ionenquellen aufgrund der gesetzlichen Bestimmungen nur bedingt einsetzbar. Die präsentierte Dielektrisch-Behinderte-EntladungsIonenquelle (DBD) kann genutzt werden, um Reaktantionen in-situ zu erzeugen, so können zum Beispiel negative Nitrationen mit einer Mischung aus Stickstoff und Sauerstoff in der DBD erzeugt werden. Die DBD Ionenquelle wird mit einer radioaktiven Ionenquelle verglichen um Aussagen über ihre Brillanz treffen zu können. Experimenteller Teil Die DBD wird mit 20 kHz und ca. 10 kV betrieben. Als Dielektrikum dient 1 mm Keramik und der Abstand zwischen der Elektrode und dem Dielektrikum beträgt 1 mm, hier brennt das Plasma. Das Plasma wird mit Stickstoff oder Sauerstoff betrieben, abhängig vom Reaktantion das erzeugt werden soll. Die Hochspannungselektrode ist in der Keramik die auch als Dielektrikum dient schlüssig befestigt. Als Gegenelektrode dient eine Edelstahlkapillare, die das Plasma von der Reaktionszone, in der die chemische Ionisation betrieben wird, trennt. Durch diese Kapillare werden die Reaktantionen in die Reaktionszone transportiert. Die Reaktionszone befindet sich in einem Ionentrichter, der bei 50 hPa betrieben wird. Nach der chemischen Ionisation werden die gebildeten Analytionen mit einem Time-of-Flight Massenspektrometer gemessen. Ergebnisse Die gute Steuerbarkeit der Chemie im Plasma, welche vom Analytgastrom getrennt brennt und eine Chemie, die prominente Reaktantionen für die negative chemische Ionsisation hervorbringt, macht die DBD zu einer vielseitigen Ionenquelle für die chemische Ionsisation. Es wird aufgezeigt, dass thermodynamisch stabile Reaktantionen wie negativ geladene Nitrat-, Carbonat-, Iodid- und Schwefelfluorid-Ionen im Plasma der DBD erzeugt werden können. Nitrationen können schon aus Spuren von Stickstoff in Sauerstoff erzeugt werden aber auch aus Spuren von Sauerstoff in Stickstoff, um die Produktion von Ozon gering zu halten. Ebenso können Carbonationen aus Spuren von Kohlenstoffdioxid in Sauerstoff erzeugt werden. Geringe Beimengungen von Iod im ppb-Bereich sorgt für die Produktion von Jodidionen. Auch Schwefelhexafluorid lässt sich im Plasma ionisieren. Zudem kann auf klebrige und korrosive Vorgängermoleküle, wie z.B. Salpetersäure zur Erzeugung von Nitrationen verzichtet werden. Dadurch kann die Chemie in der Ionenquelle relativ schnell umgestellt werden. Auch die Stabilität der Ionenquelle ist hervorragend, da die Chemie in der Ionenquelle nicht durch den Analyten gestört wird. Stärkeren Einfluss auf die erzeugte Ionenmenge hat der Gasfluss durch die DBD, dieser kann aber mit einem Flussregler stabil gehalten werden. Nach dem Einschalten der Entladung braucht es etwa 15 min bis die produzierte Ionenmenge auf einem stabilen Niveau bleibt, dieser Effekt geht vermutlich mit der Aufheizphase der Ionenquelle und der Konditionierung der Wände einher. Bisherige Vergleiche mit veröffentlichten Nachweisgrenzen (NWG) von amtosphärischen Spurengasen geben Anlass zur Annahme, dass die Kombination aus der DBD Ionenquelle und der verwendeten Transferstufe, überdurchschnittlich gute NWG erzielen. Daher soll der Vergleich einer radioaktiven Ionenquelle mit der DBD zeigen wie brillant die DBD Ionenquelle ist. Neuer Aspekte Gesundheitlich und logistisch problematische radioaktive Elemente in der Ionenquelle für die negative CI können durch eine DBD-Ionenquelle ersetzt werden.
Increasing Depth of Coverage in Data Independent Acquisition Dojahn, Joerg (1); Hunter, Christie (2); Collins, Ben (3); Gillet, Ludovic (3); Aebersold, Rudi (3) 1: AB SCIEX, Deutschland; 2: AB SCIEX, USA; 3: ETH Zurich, Zurich, SWITZERLAND Stichworte: variable windows, SWATH acquisition, ADC, TDC, detector, DIA Einleitung Data independent acquisition (DIA) strategies have been used to increase the comprehensiveness of data collection while maintaining high quantitative reproducibility. In DIA, larger fixed-width Q1 windows are stepped across the mass range in an LC timescale, transmitting populations of peptides for fragmentation, and high resolution MS and MS/MS spectra are acquired. Previous work has shown that using more narrow Q1 windows can improve peptide detection and increase sample coverage. Here both method and instrumentation advancements will be explored to continue to increase depth of sample coverage. Experimenteller Teil The MS analysis was performed on a modified quadrupole time of flight instrument equipped with an ADC detection system. DIA data collection was done using SWATH™ acquisition with prototype acquisition software to explore a variety of acquisition strategies. The DIA data was interrogated using a comprehensive yeast spectral library created from many data dependent experiments. Results assessment was performed using Excel tools. Ergebnisse Original work exploring variable window size and more narrow windows demonstrated that increasing the number of total Q1 windows from 24 to 60 windows provided an increase in confident peptide detections with good quantitative reproducibility of ~15%. To enable higher sample loads, the dynamic range of the detection system was extended by switching from a TDC based detection system to an ADC based system on a modified TripleTOF system. At the higher sample loads, we next applied increasingly narrow Q1 windows during SWATH acquisition to continue to improve the S/N in MS/MS. The number of windows was extended from 60 to 100 windows across the 400-1250 precursor m/z range while maintaining a cycle time of 3.2 secs. This provided a 20% increase in confident peptide detections with 20% or better CVs across replicates [1]. Further optimization of longer chromatography and investigations of impact on other proteomes will be discussed. Neuer Aspekte Using variable windows for SWATH acquisition Referenzen [1] Selevsek, N., Chang, C.-Y., Gillet, L.C., Navarro, P., Bernhardt, O.M., Reiter, L., Cheng, L.-Y., Vitek, O., Aebersold, R., 2015. Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH-MS. Mol Cell Proteomics mcp.M113
A targeted lipidomics approach for ceramide analysis in platelet Peng, Bing; Coman, Cristina; Ahrends, Robert Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Deutschland Stichworte: lipidomics, ceramide, LC/ESI SRM, platelet, absolute quantification Einleitung Platelets are the central building block of coagulation and hemostasis and directly linked to or affected by several metabolic diseases including diabetes, dyslipidemia and metabolic syndrome. Due to the fact that these metabolic dysfunctions might directly impact the platelet lipid composition and therefore the platelet function, it is necessary to develop a comprehensive picture of how lipids are altered and regulated in platelets. We initially focus on ceramides which are participating in the regulation of cell response such as senescence, apoptosis and autophagy [1-2]. To obtain a deep view into the ceramide lipidome we developed a selected reaction monitoring (SRM) approach to detect and quantify ceramides with high sensitivity and accuracy. Experimenteller Teil To obtain a comprehensive understanding how ceramides are regulated, it is essential to develop reliable quantification approaches. Initially, we performed lipid extraction using methyl-tert-butyl ether (MTBE) [3] to isolate lipids from human and mouse platelet pellets. Then lipid suspension was analyzed with a SRM based LC/MS lipidomics approach, which allows us to perform sensitive screening as well as absolute quantification of ceramides (spiked with internal standards) over a wide dynamic range. Ergebnisse By applying the established method, we were able to identify 160 ceramide species at fatty acid scan species level in human platelets. In mice platelets we identified around 60 ceramides species and quantified them with an excellent reproducibility over a dynamic range of 5 orders of magnitude. In these mice platelets, we achieved a coefficient of variation (CV) of 10% for technical reproducibility and a CV of 17% for the biological replicates, reflecting the good reproducibility of the ceramide analysis. When we compared the ceramide lipidome between human and mice platelets, the relative long chain base (LCB) concentration of d18:1 was the most abundant in both samples. LCB d17:0, d17:1, t17:0 and t18:0 were only found in human platelets. In general, most ceramide classes were found with similar peak area in both samples, only lactosylceramide was found less abundant in mice platelets. To test our developed approach for human platelet lipidome analysis, we compared our targeted analysis approach against a shotgun lipidomics approach. In comparison with shotgun lipidomics, more lipid species were identified in the targeted analysis LC/ESI SRM approach. In contrast, classes such as plasmalogen phosphatidylethanolamine (pPE), plasmanyl phosphatidylcholine (PCO), phosphatidic acid (PA) were only detected by shotgun analysis. Therefore, shotgun and LC based targeted lipidomics are complementing each other and should be applied both for a comprehensive lipidomics study. Neuer Aspekte A quantitative LC/ESI SRM approach for ceramides analysis at fatty acid scan species level. Referenzen [1] Münzer P, Borst. O et al. Arterioscler Thromb Vasc Biol. 2014; 34:61-71.; [2] Walter H. A. Kahr, Blood, 2009: 114 (5).; [3] Matyash V, Schwudke D et al. J Lipid Res. 2008; 49(5):1137-46.
Metabolic profiling of the battle field of competing white-rot fungi Menezes, Riya C (1); Kai, Marco (2); Svatoš, Aleš (1) 1: Max Planck Institute for Chemical Ecology, Jena, Deutschland; 2: Universität Rostock, Rostock, Deutschland Stichworte: wood-degrading fungi, pigments, secondary metabolites Einleitung Schizophyllum commune, the renowned white-rot fungus produces secondary metabolites like pigments when accosted by various white-rot fungi in nature. It can be assumed that different mycelial reactions like these can activate different genes thereby eliciting different metabolic pathways in the organism. Many microbial gene clusters may be silent under standard laboratory growth conditions. One promising approach to trigger the activation of cryptic biosynthetic pathways is the co-cultivation of two or more microorganisms in the same confined environment, leading to the production of new metabolites. Such compounds, produced dynamically, are of potential interest as new leads for drug discovery. Thus, this study focuses on metabolites produced by S. commune during interspecies crosstalk with wood-decay fungi - Hypholoma fasciculare, and Pleurotus ostreatus. Experimenteller Teil To investigate the fungal cocultures, agar plate based confrontation assays were performed between S. commune and, H. fasciculare and P. ostreatus. From the co-cultured plates, mycelial strips were cut out from the interaction zone and from the periphery of fungi , respectively. Each sample was subjected to a methanol:ethyl acetate extraction and finally diluted in methanol. UHPLC–ESI–MS/MS was performed with the diluted extract. Full scan mass spectra were generated using 30,000 m/Δm resolving power. PCA was performed on the full-scan data of the samples using Metaboanalyst 3.0, a web-based tool for metabolomic data processing, statistical analysis, and functional interpretation. Ergebnisse In self-paired cultures the fungi did not exhibit induction of pigmentation, while coloured substances and discolouration of the medium was observed when S. commune interacted with H. fasciculare and P. ostreatus. A greenish blue/sometimes black pigment developed at the bottom of the plate within the domain occupied by S. commune at 12 h postcontact. The intensity of this pigmentation increased with the duration of contact and was visible in the interaction zones of the fungi. LESA-HRMS was performed on the confrontation assay plates directly from the surface of the agar. Analyzing the interaction zone, a mass signal corresponding to the pigments indigo, indirubin and isatin were observed. Statistical analyses of the different zones of the fungal cocultures showed a distinction between metabolites produced by S. commune interacting with H. fasciculare and P. ostreatus. There was also a difference noted in the mycelial metabolites from those that were secreted. These patterns suggest that in S. commune different effectors are elicited in response to different competitors. In order to allow scientists observations of natural phenomena in the most direct and non-invasive ways, new tools have constantly been developed. Because of the complexity of microbial extracts, advanced analytical methods (e.g., mass spectrometry based metabolomics) are the key for the successful detection and identification of co-cultureinduced metabolites. Thus, we showed that a direct technique like LESA and an indirect one like LC-MS are efficient tools to identify compounds which have a relatively low solubility in water and organic solvents, necessitate only low invasion and are label-free applications requiring minimal sample preparation. Therefore, they are well suited for biological applications. Neuer Aspekte The techniques open up avenues for investigations of fungal interactions with co-occurring organisms to obtain information on their mechanisms Referenzen
[1] Menezes RC, Kai M, Krause K, Matthäus C, Svatoš A, Popp J & Kothe E (2014). Anal Bioanal Chem 10.1007/s00216-014-8383-6;
DOI
[2] Kertesz V, Van Berkel GJ (2010) Fully automated liquid extraction based surface sampling and ionization using a chip-based robotic nanoelectrospray platform. J Mass Spectrom 45(3):252–260; [3] Boddy L (2000) Interspecific combative interactions between wood decaying basidiomycetes. FEMS Microbiol Ecol 31(3):185–194; [4] Xia J, Mandal R, Sinelnikov IV, Broadhurst D, Wishart DS (2012) MetaboAnalyst 2.0—a comprehensive server for metabolomic data analysis. Nucleic Acids Res 40:W127–133, Web Server issue
Online Photostability Study of Pharmaceutical Substances Zulkiewicz Gomes, Diego (1,2); Assaf, Jaber (1); Schulze, Thomas (1); Parr, Maria Kristina (1) 1: Freie Universität Berlin, Deutschland; 2: IPT – Institute for Technological Research, Brazil Stichworte: photostability, product identification, reduction of animal studies, kinetics Einleitung Study of the photostability of pharmaceutical substances is becoming increasingly important in drug development. Thus pharmaceutical industries are also requested by the regulations agencies to provide data for new or renewed licences of medicines. The aim of this work is to evaluate the photostability of drug substances by using a modified HPLC, which consists of a tailored online photoreactor with back-flush and two column system. Coupling to a mass selective detector allows to identify the newly formed photoproducts. By this method, it is possible to obtain a straightforward answer on the degradation via photolysis of different substances in-vitro and to predict the photostability in early stage of drugs development. This may also help to reduce animal experiments in the future. Experimenteller Teil Ketoprofen aqueous solution (pH 7.0) was irradiated with UVA during diferent periods of time. The peaks were colected and analysed by LC-MS single quadrupole and also by ESI-TOF. The photoreactor consist of a UVA lamp surrounded by a braided and twisted capillary inside an Alumminum tube. The temperature is also controled and monitored during all the experiment. After the substances run throught the photoreactor and the degradation products are formed, they are first trapped in a Aqua C18 column and then eluted in reverse flow in a second column (Onyx monolythis C18). Ergebnisse Irradiation of the nonsteriodal anti-inflamatory agent ketoprofen with UVA resulted in decreasing amounts of the target compound. First order kinetics was found as best model. Several photoproducts were detected after irradiation in aqueous solution (pH 7.0),when maintaining the temperature in the reactor at 25-30°C. Mass spectrometric characterization supported the structures reported from literature. These data showed the potential for the use of the new device for fast and easy photostability studies, that may help to reduce time consuming in-vitro experiments and animal trials. Targeted MS/MS-based methods may be generated using the results obtained by this online-irradiations for use in-vitro and in-vivo. Scale-up may also be realized for the generation of reference material for quantification as well as for toxicity testing. Neuer Aspekte A new online-hν-HPLC device is presented that allows easy study of photodegradation and direct coupling to MS(/MS) for product identification.
To which extent are oxidized phospholipids digestible by the enzyme phospholipase A2? - A mass spectrometric and NMR spectroscopic study Schröter, Jenny; Kristin, Zschörnig; Beate, Fuchs; Rosemarie, Süß; Jürgen, Schiller University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics Stichworte: MALDI-TOF MS, ESI MS, Lipid Oxidation, Phospholipase A2. Einleitung Free fatty acids are released through phospholipase A2 (PLA2), which cleaves the fatty acyl residue in sn-2 position of the corresponding phospholipid (PL) [1]. During inflammatory diseases, reactive oxygen species (such as HOCl) lead to the formation of oxidatively modified PLs (e.g. chlorohydrin generation). Oxidation of PLs may significantly influence their digestibility through PLA2. Furthermore, the position of the unsaturated fatty acyl residue (sn-1- or sn2-position) and the modification of the headgroup (phosphatidylcholine (PC) versus phosphatidylethanolamine (PE)) may lead to major differences with respect to the PLA2 activity. Therefore, the aim of this study is to investigate these aspects using a combination of MALDI-TOF and ESI-IT MS as well as high resolution NMR and HPTLC. Experimenteller Teil All chemicals, solvents, PLA2 and the applied MALDI matrices (9-aminoacridine and 2,5-dihydroxybenzoic acid) were obtained from Sigma-Aldrich. Stock solutions of PLs were purchased from AVANTI Polar Lipids and used as supplied. The PL oxidation was performed as recently described [2,3] using either HOCl or the Fenton system (Fe2+/H2O2). The oxidized PLs were enriched by extraction with organic solvents and chromatographically purified. MALDI-TOF and ESI-IT mass spectra were recorded on a Bruker Autoflex and Bruker Amazon, respectively. HPTLC experiments were performed applying unmodified silica gel (Merck) and chloroform, ethanol, water, and triethylamine (35:35:7:35, v/v/v/v) as mobile phase. High resolution NMR spectra were recorded either on a Bruker AMX 300 or AVANCE 600 MHz spectrometer. Ergebnisse Isolated standards of PC (PC 16:0/18:2 (PLPC)) as well as two PEs (PE 16:0/16:0 (DPPE) and 16:0/18:2 (PLPE)) with defined fatty acyl compositions were oxidized with HOCl under identical conditions to enable the direct comparison of the oxidative effect of HOCl regarding the unsaturated fatty acyl residues and - in the case of the PEs - the PL headgroup, too. All investigated, unsaturated PLs react readily with HOCl. Using a sufficient excess of HOCl, PLPC could be quantitatively converted into the corresponding dichlorohydrin if the reaction was performed in absence of chloride. There were only minor amounts of the corresponding lysophosphatidylcholine (LPC 16:0), i.e. a hydrolytic cleavage does not occur. Whereas the unmodified PLPC can be easily digested by PLA 2, all used analytical methods clearly indicate (with the aid of the PC/LPC ratio) that the corresponding chlorohydrin product is digested less efficiently. However, digestion products of the chlorohydrin modified PLPC are detectable using a higher enzyme concentration. Thus, inactivation of PLA2 is mediated by the chlorohydrin product of PLPC. Similar data were obtained if chain-shortened oxidation products of PLPC (such as 1-palmitoyl-2-(9-oxo-nonaoic acid)-snphosphatidylcholine) or hydroperoxides of PLPC were investigated. Compared to the studied PC, the investigations of PEs regarding their oxidation capability are much more complex. PEs can be modified at both the (unsaturated) fatty acyl residues and the (amine) headgroup. For instance, the presence of HOCl leads to the formation of mono- and dichloramines, which are rapidly converted into other products such as the corresponding aldehydes, nitriles or imines. Hence, the examinations of these products are significantly aggravated by the cross reactivity of the amino group. According to our current data, however, modifications of the headgroup are less important in comparison to the fatty acyl residues that agree with the specificity of the enzyme PLA2. Neuer Aspekte The reduced digestibility of oxidized phospholipids by PLA2 was proven by different MS and spectroscopic methods. Referenzen
[1] Fuchs B, Müller K, Paasch U, Schiller J (2012) Lysophospholipids: potential markers of diseases and infertility? Mini-Rev Med Chem. 12:74-86.; [2] Zschörnig K, Schiller J (2014) A simple method to generate oxidized phosphatidylcholines in amounts close to one milligram. Chem Phys Lipids 184C:30-37.; [3] Arnhold J, Osipov AN, Spalteholz H, Panasenko OM, Schiller J (2002) Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid. Biochim Biophys Acta 1572:91-100.; [4] Richter G, Schober C, Süss R, Fuchs B, Birkemeyer C, Schiller J (2008) Comparison of the positive and negative ion electrospray ionization and matrix-assisted laser desorption ionization-time-of-flight mass spectra of the reaction products of phosphatidyl
Lipidomics of Alzheimer’s Disease using an Integrated Microfluidic-Ion Mobility-MS Device Weibchen, Gunnar (1); Lai, Steven (2); Heywood, David (2); Doneanu, Angela (2); Murphy, Jim (2); Langridge, James (3); Astarita, Giuseppe (2) 1: Waters GmbH, Deutschland; 2: Waters Corporation, Milford, MA, USA; 3: Waters Corporation, Manchester, UK Stichworte: Metabolomics, Lipidomics, Lipid, Ion Mobility, Alzheimer’s disease Einleitung Alzheimer's disease (AD) is the most common cause of adult dementia, but the cause of this inexorable neurodegenerative disease remains still elusive. Alterations in lipid pathways have been associated with AD. Here we used a novel MS device to conduct an untargeted lipidomic investigation of AD human tissues. Experimenteller Teil A microfluidic-ion mobility-MS device was optimized for MS analysis of lipids in complex biological extracts. The integrated microfluidic device was fabricated from resistant ceramic materials that permit operation at high pressure with sub 2 µm particles, leading to highly efficient LC separations of lipid molecules. Lipids were separated using 150 µm ID x 100 mm devices packed with reversed phase C18, 1.7 µm particles at flow rates of 3 µl/min. By integrating microscale LC components into a single platform design, the devices avoided problems associated with capillary connections and the need to keep the system free of leaks, blockages, and excessive dead volume. Such an integrated microfluidic-ion mobility-MS device had a performance comparable to analytical scale LC-MS analysis. Ergebnisse We used an integrated microfluidic-ion mobility-MS device to survey frozen tissue samples from clinically characterized AD patients and age-matched controls. Mobile phases and analysis times were similar to regular LC methods using analytical-scale columns. Data was collected using both negative and positive mode in the dataindependent acquisition mode with an alternate low and elevated collision energy method to acquire both precursor and product ion information in a single analytical run. Lipids profiles were processed using multivariate and patternrecognition tools to group the observed changes in lipids, which were identified using online database search. Travelling-Wave ion mobility was integrated into the novel microfluidic-MS device to give improved peak capacity and CID fragmentation specificity. Ion mobility-derived collision cross sections provided orthogonal physicochemical data that were used with retention time, accurate mass and MS/MS data to increase confidence of lipid identification. The information obtained was integrated with clinical data to generate testable hypotheses on the functional significance of the lipid abnormalities observed in AD. Neuer Aspekte Microfluidic-ion mobility-MS-based lipidomics reveals novel molecular alterations in Alzheimer’s disease.
The Use of Fragment Ion and Collision Cross Section for Confident Identification from LC-Ion Mobility-MS Metabolomics Data Weibchen, Gunnar (1); Isaac, Giorgis (2); Astarita, Giuseppe (2); Lai, Steven (2); Ladak, Adam (2); Langridge, James (4); Shockcor, John (2); Borthwick, Andy (3) 1: Waters GmbH, Deutschland; 2: Waters Corporation, Milford, MA; 3: Nonlinear Dynamics, Newcastle, UK; 4: Waters Corporation, Manchester, UK Stichworte: Metabolomics, Lipidomics, ion mobility, collisional cross-section (CCS) Einleitung Mass spectrometry based technologies often couple with UPLC-Ion Mobility-MS to measure the level and variation of metabolites in biofluids. Data generated through metabolomics studies can yield important metabolic insights into disease onset and progression. The identification of unknown compounds is often a bottleneck in large scale metabolomics profiling. UPLC-Ion Mobility-MS based metabolomics generate large and complex datasets and data analysis, interpretation and identification is the slowest step in any metabolomics workflow. In this study, we introduce a simple and novel bioinformatics tool that has been specifically developed for the large scale analysis of UPLC-Ion Mobility-MS data for confident identification of small molecules based on fragment ion and collisional cross-section. Experimenteller Teil Urine samples were collected from a single healthy individual. The urine was centrifuged and 600µL of the urine supernatant was taken and dilutes with 1800µL of water. The urine was divided into three groups: control, low dosed (LD) and high dosed (HD). To create a working differential urine sample 11 different drugs were spiked to the LD and HD urine. A simple gradient elution at a flow rate of 500 µL/min was applied with a mobile phase consisting of water and acetonitrile both in 0.1% formic acid on a reversed phase AQUITY column (HSS T3, 2.1x100 mm, 1.8 µm). The data were acquired on SYNAPT G2-S HDMS™ system operated in positive mode using the MSE or HDMSE data acquisition technique. Ergebnisse The software adopts an intuitive workflow approach to performing comparative UPLC-Ion Mobility-MS metabolomics data analysis. The workflow starts with UPLC-Ion Mobility-MS raw data file loading then retention time alignment and deconvolution, followed by analysis that creates a list of features. The features are then identified based on exact mass, MS/MS fragment, isotope distribution, retention time and collisional cross-sectional area (CCS). A simple dataset consisting three sample mixtures was created to illustrate the processing and identification of features. A control urine (no spiking), LD urine (spiked with 4 LD drugs) and HD urine (spiked with 4 HD drugs). According to the experimental design the software should: Identify all the spiked standards, classify three distinct groups and show high level of relative abundance of the spiked standards for the LD and HD groups compared to control. To improve the confidence in the compound identification theoretical fragmentation of a candidate list of compounds and their calibrated CCS was used. The resulting ‘in silico’ fragmentation was matched against the measured/observed fragments and CCS of the compound database. For the in silico fragmentation, the candidate molecules are selected from a compound database based on the exact mass. Using this list of candidates the fragmentation algorithm generates all possible fragments for a candidate compound in order to match the fragment mass with the measured peaks. The identification data can be stored and used to perform fragment and CCS database searches. This allows building custom databases of fragment information, CCS and retention time which can then be used to perform future identifications when using the same compound database. The results showed that the spiked standards were identified as top rank based on MS/MS fragment and CCS compared to identification based on retention time and exact mass. The functionality of the software will be demonstrated using biological samples. Neuer Aspekte A novel and confident identification based on fragment ion and CCS of UPLC-Ion Mobility-MS data from metabolomics and lipidomics datasets.
Unravelling 2-deoxy-2-fluoro-D-glucose metabolism in plant tissue using mass spectrometry and NMR Fatangare, Amol (1); Paetz, Christian (2); Saluz, Hans Peter (3); Svatos, Ales (1) 1: Mass spectrometry and proteomics research group, Max Planck institute for chemical ecology, Jena, Germany; 2: Biosynthesis / NMR research group, Max Planck institute for chemical ecology, Jena, Germany; 3: Cell and molecular biology department, Hans Knoll institute, Jena, Germany. Stichworte: Arabidopsis thaliana, FDG (2-deoxy-2-fluoro-D-glucose) metabolism, Mass spectrometry, NMR, Fmaltose. Einleitung 2-Deoxy-2-Fluoro-D-glucose (FDG) is a structural glucose analogue commonly used as a radioactive glucose surrogate in clinical diagnostics and animal studies. It mimics the glucose distribution and it was assumed that after uptake, it is metabolized via glycolysis pathway to FDG-6-phosphate but not further[1]. However, numerous papers describe the fate of FDG to FDG-6-P and further metabolites in the animal cells [2]. FDG has also been employed in plant radiotracer studies but its metabolism in plant cells is not yet characterized. Here, we applied FDG to Arabidopsis leaf and analyzed leaf extract for fluorine (19F) metabolites using MS and NMR. We demonstrate that FDG metabolism in plant cells is considerably different than animal cells and goes beyond FDG-6-P. Experimenteller Teil Mature leaves of A. thaliana (short day plants, 6-7 week, early flowering stage) were gently pricked on the abaxial surface. Five microliter of FDG (20 mg.mL-1) was immediately applied in the pricked spots. Four hours later leaves were extracted using Chloroform:Methanol:Water (1:2:1). Aqueous fraction was analyzed by LC-MS/MS and NMR for the presence of 19F-containing compounds. Ergebnisse Elucidating FDG metabolism in plants is a crucial aspect for establishing its application as a radiotracer in plant imaging. Here, we describe the metabolic fate of FDG in model plant species, Arabidopsis thaliana. LCMS and direct infusion MS results confirmed the presence of 5 different 19F containing metabolites in the extract. In total, we putatively identified above 19F containing metabolites as FDG (m/z 181.0513), F-gluconic acid (m/z 197.0464), FDG6-P (m/z 261.0180), F-maltose (m/z 343.1051), and UDP-FDG (m/z 567.0434) on the basis of known literature information, their exact mono-isotopic mass (± 5 ppm mass error) and MS/MS fragmentation analysis. Characterization of purified compounds using NMR confirmed identification of 19FDG-6-P (m/z 261.0180), and 19Fmaltose (m/z 343.1051) as major end products of 19FDG metabolism in A. thaliana leaf cells. Neuer Aspekte Elucidation of FDG metabolic fate in plant cells. 19F-maltose biosynthesis Referenzen [1] 1. Phelps, M.E. Molecular imaging with positron emission tomography. Annual Review of Nuclear and Particle Science. 2002; 52: 303-338.; [2] 2. Kaarstad,K. Bender,D.,Bentzen, L., Munk,O.J. and Keiding, S . Metabolic Fate of 18F-FDG in Mice Bearing either SCCVII Squamous cell Carcinoma or C3H Mammary Carcinoma. J. Nuvlear Med. 2002; 43.
Synthese und massenspektrometrische Charakterisierung von Aib-haltigen antibiotischen Peptiden Laub, Annegret Leibniz-Institut für Pflanzenbiochemie, Deutschland Stichworte: Festphasenpeptidsynthese, UHPLC-ESI-HR-Tandem-Massenspektrometrie von Peptaibolen Einleitung Peptaibole sind Sekundärmetaboliten von parasitären Pilzen, die sich durch ein interessantes biologisches Wirkspektrum auszeichnen [1]. Dabei handelt es sich um Peptide, die durch hohe Gehalte nicht-proteinogener Aminosäuren charakterisiert sind. Die dadurch verursachte Bildung helikaler Sekundärstrukturen ist für die membranmodifizierenden Eigenschaften der Peptaibole verantwortlich [2]. Durch die Entwicklung von Synthesestrategien zur Totalsynthese von Naturstoffen können die so dargestellten Peptaibole als Referenzsubstanzen für die Strukturaufklärung von zuvor isolierten Verbindungen sowie zu umfangreichen biologischen Untersuchungen genutzt werden [3, 4]. Experimenteller Teil Die Festphasenpeptidsynthese der Peptaibole wurde mittels Fmoc-Strategie automatisiert in einem Peptidsynthesizer ResPep SL (intavis Bioanalytical instruments) durchgeführt. Die abschließende N‑Acetylierung wurde an dem festphasengebundenen Reaktionsprodukt manuell vorgenommen. Die Aufreinigung erfolgte mittels Säulenchromatographie sowie präparativer HPLC. Die Charakterisierung der dargestellten Verbindungen wurde mittels ESI-HR-MSn unter Verwendung von stoßinduzierten Dissoziationsmassenspektren (CID, collision induced dissciation) im positiven und negativen Ionenmodus mit einem Orbitrap Elite Massenspektrometer (Thermofisher Scientific) durchgeführt. Die chromatographische Trennung der zu untersuchenden Probenlösung erfolgte mittels UHPLC (Dionex UltiMate 3000, Thermo Scientific) mit einer RP-C18-Säule (Hypersil GOLD; Thermo Scientific). Als mobile Phase wurden ein Gemisch aus Wasser und Acetonitril (jeweils mit 0.2% Ameisensäure) unter Verwendung eines Gradientensystems verwendet. Ergebnisse Im Rahmen dieser Arbeit war es möglich, die Primärstruktur der bislang unbekannten Chilenopeptine A und B und der Albupeptine A und C mit Hilfe von UHPLC‑ESI-HR-MSn-Messungen im negativen sowie positiven Modus aufzuklären und somit die vorgeschlagenen Sequenzen der nativen Strukturen zu bestätigen. Durch den Einsatz des enantiomerenreinen D-Isovalins (D-Iva) bei der Synthese der Albupeptine A und C konnte die Zuordnung der absoluten Konfiguration des Isovalins (D-Iva) in dieser Verbindungen mittels NMR bestimmt werden. Am Beispiel des Chilenopeptins A wird das unterschiedliche Fragmentierungsverhalten der [M+H] +- und [M+2H]2+-Ionen im MS²Modus diskutiert. Bei den [M+H]+-Ionen bilden sich im MS² Fragmente der b-Reihe. Im Gegensatz dazu weist das [M+2H]2+-Ion im MS²-Spektrum zwei charakteristische Fragmente auf, die aus dem Bruch der labilen Bindung zwischen Aib-Pro resultieren. In diesem Fall werden insbesondere ein N-terminales b-Fragment- und ein C‑terminales y-Ion beobachtet. Neuer Aspekte Strukturaufklärung und Totalsynthese neuer Peptaibole aus chilenischen Pilzen Referenzen [1] Degenkolb T, Kirschbaum J, Brückner H (2007): New Sequences, Constituents, and Producers of Peptaibiotics: An Updated Review. Chem. Biodiv. 4,1052–1067.; [2] Aravinda S, Shamala N, Balaram P (2008): Aib Residues in Peptaibiotics and Synthetic Sequences: Analysis of Nonhelical Conformations. Chem. Biodiv. 5, 1238–1262.; [3] Hjørringgaard CU, Pedersen JM, Vosegaard T, Nielsen NC, Skrydstrup T (2008): An Automatic Solid-Phase Synthesis of Peptaibols. J. Org. Chem. 74, 1329–1332.;
[4] Degenkolb T, Berg A, Gams W, Schlegel B, Gräfe U (2003): The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions. J. Pept. Sci. 9, 666– 678.
Advances in Native Mass Spectrometry-based methods for the analysis of Non-Covalent Protein complexes Williams, Jonathan P. Williams (2); Kipping, Marc (1); Anderson, Malcolm (2); Jackson, Lidia (2); Giles, Kevin (2); Brown, Jeff (2) 1: Waters GmbH, Deutschland; 2: Waters Corporation, Manchester, UNITED KINGDOM Stichworte: Intact, Protein, Complexes, Non-covalent, ETD Einleitung Native Mass Spectrometry involves the generation, transmission and detection of intact non-covalently assembled protein-protein and protein-ligand complexes which often yields a narrow charge-distribution at high m/z predominantly using nano-ESI. The high m/z capability of time-of-flight mass analysers has made them the instrument of choice for the study of such complexes. Here, we investigate the high m/z characteristics of a quadrupole/ion mobility/time-of-flight instrument (Q-IM-TOF) incorporating a dual stage conjoined source ion guide for the transmission and subsequent fragmentation using CID and/or ETD of typical assemblies. We will present examples such as human haemoglobin (64 kDa), ADH (147 kDa), GDH (335 kDa) and GroEL (802 kDa). Size exclusion UPLC-MS was also investigated using non-denaturing conditions in order to preserve the native complexes. Experimenteller Teil A Q-IM-TOF mass spectrometer (Synapt G2-S) was operated in both CID and ETD modes. ETD was performed in the trap travelling wave ion guide (containing He) and CID was effected by raising the ions energy as they entered the trap or transfer ion guide (containing Ar). Direct infusion was conducted by nanoESI using pre-cut PicoTip emitters. SE-UPLC made use of an Acquity UPLC I-Class Bio System with a BEH200 SEC 1.7 µm, 4.6 x 150 mm column. The complexes were injected into an aqueous mobile phase containing 10mM Ammonium Formate at a flow rate of 150μL/min. Ergebnisse We will present data detailing the extent to which ETD is a valuable additional and complementary fragmentation technique to CID for such species, facilitating extensive cleavage of the protein backbone of the sub-units from within the homo- and hetero-complexes. For the nano-ESI infusion studies which combined CID and ETD, the protein complexes were infused separately. The ETD of human haemoglobin will be shown to have the potential for phenotyping human hemoglobinopathies. Moreover, the applied on-line SE-UPLC MS separation technique has significant potential for future top-down MS studies of proteins in their denatured or native states whilst offering a desalting step prior to MS. Neuer Aspekte Novel use of ETD using Synapt G2-S for potential surface mapping and phenotyping of protein complexes in their native state.
Bacteriophage endolysin activity is modulated by quaternary state. Krichel, Boris (1); Dunne, Matt (2); Meijers, Rob (2); Uetrecht, Charlotte (1) 1: Heinrich Pette Institut, Deutschland; 2: European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, DE69117 Heidelberg, Germany. Stichworte: Virology, Native Mass Spectrometry, Biotechnology Einleitung Endolysins are produced in the final stage of bacteriophage infections to lyse bacterial peptidoglycan causing the cell wall to rupture killing the bacterial host cell. The timing of lysis is strongly regulated and is thought to involve a molecular switch. Experimenteller Teil Here three bacteriophage endolysins were studied with focus on quaternary structure influencing lytic activity. We found that free C-terminal domain (CTD) plays an essential role for endolysin higher order assemblies. Native mass spectrometry (MS) was used to analyze subunit stoichiometry of intact endolysins in vacuo and to determine ratios of different species in solution. By this means coexisting oligomeric states of wild type endolysins were identified and confirmed after CID (collision induced dissociation). The distribution of species was then compared to lytic activity. Ergebnisse The most abundant oligomer, a 2:2 hetero-tetramer consisting of the CTD and full length protein, is likely the most active oligomeric species. Mutants affecting the CTD production relative to the full length protein influence the oligomeric state of the endolysin and reduce lytic activity. In order to restore activity His-tagged CTD was titrated to monomeric full length protein and analyzed via native MS and in a lysis activity assay. Up to a threefold molar excess of CTD the lysis activity increases carried mainly by the 2:2 and 3:1 hetero-tetramer. However, full wild type activity was never recovered. These results demonstrate how enzymatic activity is regulated by switching the relative abundance of differently active protein complexes. Neuer Aspekte Secondary translation of endolysin subunit via ribosomal binding site Relation between oligomeric state and lytic activity
Thermal/optical Carbon Analysis coupled with Photoionization Time-of-Flight Mass Spectrometry: Fine Particulate Matter from a Marine Engine Czech, Hendryk (1); Sippula, Olli (2,3); Sklorz, Martin (1); Streibel, Thorsten (1,3); Zimmermann, Ralf (1,3) 1: Joint Mass Spectrometry Centre University of Rostock, Chair of Analytical Chemistry, Germany and HelmholtzZentrum München, CMA (Comprehensive Molecular Analytics), Germany; 2: University of Eastern Finland, Department of Environmental Science, Fine Particle and Aerosol Technology; 3: Helmholtz Virtual Institute of Complex Molecular Systems in Environmental Health (HICE) - www.hice-vi.eu Stichworte: single-photon ionization (SPI), resonance-enhanced multiphoton ionization (REMPI), hyphenation, fine particulate matter (PM), marine engine Einleitung Worldwide 90% of goods are transported by ships, mainly operating on heavy fuel oil (HFO) with an average fuelsulfur content of 2.7%. Thereby, several million tons of fine particulate matter (PM) are emitted per year, composed of soot, metal oxides, sulfates and nitrates, but also organics adsorbed on particle surface [1]. Previous studies identified ship-related PM as a perpetrator of cardiovascular diseases and lung cancer [2]. Furthermore, it affects the radiative forcing of the Earth depending on its composition [3]. In 2020, the legal fuel-sulfur content for ships will be statutorily restricted to 0.5% in coastal areas or even to 0.1% in sulfur emission control areas (SECA). Therefore, the qualitative and quantitative changes in emission have to be evaluated. Experimenteller Teil PM of HFO and diesel fuel (DF) from a marine engine was collected on quartz fiber filters and analyzed by a thermo/optical carbon analyzer (TOCA) which was coupled to a time-of-flight mass spectrometer (TOFMS) using single photon ionization (SPI) at 118 nm and resonance-enhanced multi photon ionization (REMPI) at 266 nm [4]. Among the quantification of organic (OC) and elemental carbon (EC) by “ImproveA” temperature protocol, approximately 20% of the evaporating compounds are alienated to the TOFMS, which allows the detection of molecular ions of single compounds in thermodesorption-like (25°C – 280°C) and pyrolysis-like fraction (280°C – 580°C). REMPI refers to a selective ionization technique for aromatics, whereas SPI is regarded as a more universal technique of soft ionization. Ergebnisse HFO-derived particles contained the triple quantity of OC (38.6 mg/m³) compared to DF-derived particles (14.3 mg/m³), but approximately the same quantity of EC (HFO: 8.0 mg/m³; DF: 8.6 mg/m³), which finds also expression in the different ratios of OC/EC (LFO: 4.8; HFO: 1.8). In the thermodesorption-like OC-fraction of DF, the mass spectrum is dominated by methyl esters of fatty acids, such as oleic acid, linolic acid and palmitic acid, which are known as biodiesel components. The thermodesorption-like OC-fraction of HFO showed mainly alkylated dibenzothiophenes and phenanthrenes along with other low-weight polycyclic aromatic hydrocarbons (PAH). In the pyrolysis-like OC-fractions some similar thermal fragments occurred for both fuels. Sulfur-containing thermal fragments as well as large PAH seems to be characteristic for HFO. This observation was examined more in detailed by running a modified Student’s t-test [5] for the mass spectra of both fuels and the respective thermal fraction. It turned out that alkanes, (low-alkylated) 2- to 4-ring PAH and corresponding oxy-PAH were significantly enhanced on the DF-particles, whereas HFO-particles contained significantly more n-ring-PAH with n ≥ 5 and PAH with a high degree of alkylation. Moreover, the combustion was investigated by comparing ratios of parent and alkylated PAH in fuel and particulate emission. Parent PAH are mainly formed by flame synthesis while alkylated PAH in the emission indicate unburned fuel. Compared to the fuel, diesel fuel particles are enriched in parent PAH, but for HFO the distribution of alkylation remains constant. Hence, two different fuel-dependent combustion processes can dominate in the same engine leading to different type of PAH with different toxicity equivalent factor (TEF). Thus, toxicological studies must follow to assess whether the potential change in effect of ship-related PM on human health. Neuer Aspekte
PM from a marine engine was analyzed by thermal/optical carbon analysis coupled to time-of-flight mass spectrometry with photoionization. Referenzen [1] J. Moldanová et al., Atmospheric Environment 43, 2632 (2009).; [2] J. J. Corbett et al., Environmental Science and Technology 41, 8512 (2007).; [3] A. Lauer et al., Atmospheric Chemistry and Physics 7, 5061 (2007).; [4] J. Grabowsky et al., Analytical and Bioanalytical Chemistry 401, 3153 (2011).; [5] J. B. D. V. Weir, Nature 187, 438 (1960)
SpheriCal® - Monodisperse Polyester Dendrimers as Universal Mass Calibrants in Mass Spectrometry Boertz, Jens (1); Grayson, Scott M. (2); Malkoch, Michael (3) 1: Sigma-Aldrich, Fluka, Buchs SG, Switzerland; 2: Tulane University, Department of Chemistry, New Orleans, USA; 3: Polymer Factory Sweden AB, Stockholm, Sweden Stichworte: SpheriCal, monodisperse, mass calbration Einleitung Proteins or peptides were an obvious choice to meet the need for calibrants during early mass spectrometry studies of high mass ranges (m/z > 1000) because these large biomolecules were widely available and monodisperse. However, they typically exhibited poor stability, and therefore required care during storage and sample preparation. While traditional linear polymers within a similar mass range can be easily prepared with enhanced stability, they exhibit polydispersity which can complicate accurate mass identification. Dendrimers, however, are a class of synthetic macromolecules that are prepared using an iterative step-wise synthesis, and therefore offer high molecular weights and monodispersity in addition to chemical robustness. A family of polyester dendrimers have been prepared and evaluated for use as calibrants. Experimenteller Teil A library of polyester dendrimers were prepared according to previously reported techniques with molecular weights ranging from 700 to 30000 u. MALDI-TOF-MS was used to characterize crude reaction mixtures to verify that each step in the synthesis was driven to completion, and to confirm the monodispersity of the product after workup. MALDI-TOF-MS sample preparation utilized 9-nitroanthracene as the matrix, sodium trifluoroacetate as the counter ion and tetrahydrofuran as the solvent. All chemicals and solvents were purchased from Sigma-Aldrich and used without further purification. Mass spectrometric analysis was performed using a Bruker Autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA.) Data was acquired using reflector-positive ion mode, with an acceleration voltage of 20 kV, and delayed extraction.
Ergebnisse The dendrimer calibrants exhibited a number of attractive advantages, including exceptional shelf-lives, broad compatibility with a wide range of matrices and solvents, and evenly spaced calibration masses across the mass range examined, 700-30,000 u. The exceptional purity of these dendrimers and the technical simplicity of this calibration platform validate their broad relevance for high molecular weight mass spectrometry. Neuer Aspekte Monodisperse dendrimers exhibit enhanced shelf-life and compatibility with matrix and counterion enhancing their utility as calibrants. Referenzen [1] Ihre, H.; Padilla de Jesus, O. L.; Fréchet, J. M. J. J. Am. Chem. Soc. 2001, 123, 5908-5917; [2] Malkoch, M.; Malmström, E.; Hult, A. Macromolecules 2002, 35, 8307–8314; [3] Grayson, Scott M.; Myers, Brittany K.; Bengtsson, Jonas; Malkoch, Michael J. Am. Soc. Mass. Sprectrom. 2014, 25, 303-309
SILAC-Based Secretome Analysis of Non-Small Cell Lung Cancer Cell Lines Bosse, Konstanze (1); Haneder, Silvia (2); Ihling, Christian (1); Seufferlein, Thomas (3); Sinz, Andrea (1) 1: Martin-Luther-Universität Halle, Universitätsklinikum Ulm, Deutschland
Deutschland;
2:
Spherotec
GmbH
Martinsried,
Deutschland;
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Stichworte: SILAC, NSCLC, secretome, cancer Einleitung Lung cancer is the most common cancer fatality in Europe (335,000 deaths/year) [1] with non-small cell lung cancer (NSCLC) comprising 80-85% of all cases [2]. Survival rates are poor (5-year survival rate lower than 15%) as most patients develop a metastatic disease that is too advanced for curative surgical resection. Even for these patients who undergo surgery, cure is not guaranteed. Tumor-host interaction plays a major role in carcinogenesis, but also in resistance to apoptosis- and necrosis-inducing treatment strategies. This interaction is modified by therapeutic interventions and changes substantially when tumor cells become resistant to a therapeutic agent. Analyzing the secretome of NSCLC cells may provide novel targets to overcome the therapeutic resistance of lung cancer. Experimenteller Teil For quantitative comparison of proteins secreted by two different NSCLC cell lines the SILAC (stable isotope labeling with amino acids in cell culture) approach was applied. Mixed SILAC cell supernatants were concentrated via ultrafiltration. After SDS-PAGE followed by tryptic in-gel digestion the resulting peptide mixtures were separated by nano-HPLC (UltiMate, Dionex) and analyzed by nano-ESIMS/MS (LTQ-Orbitrap XL, Thermo Fisher Scientific). Eight biological replicates were performed. Differentially regulated proteins were evaluated based on their heavy/light SILAC ratios, the number of unique peptides, and their origin as secreted proteins. The interactions of selected proteins will be further investigated by chemical cross-linking combined with mass spectrometry to identify binding partners and its interface regions. Ergebnisse To differentially quantify NSCLC secretomes, a stable isotope label can in principle be introduced by chemical modification or by metabolic labeling [3]. The earliest stage for introducing stable isotope signatures into proteins is by metabolic labeling during cell growth and division [4]. SILAC was chosen as it is a metabolic labeling approach [4]. Heavy (13C-arginine and 13C-lysine labeled) and light (12C-arginine and 12C-lysine labeled) states of NSCLC cell lines PC9 (erlotinib sensitive) and PC9ER (erlotinib resistant) were analyzed. On average, 900 proteins were identified in each sample with low variations in the numbers of identified proteins, indicating the robustness of our approach. Applying abovementioned criteria to identify protein candidates that are differently regulated between PC9 and PC9ER cells yielded 17 proteins, with five proteins (tissue-type plasminogen activator, epidermal growth factor receptor, urokinase-type plasminogen activator, platelet-derived growth factor d and stromal cell-derived growth factor) showing the most prominent different regulation. Tissue-type plasminogen activator (t-PA) is an already established tumor marker for other cancer types and seems to be a promising biomarker for erlotinib resistance in NSCLC cells. Therefore different variants of t-PA were successfully expressed in E. coli BL21(DE) and purified by immobilized metal ion affinity chromatography (IMAC). As a next step, cross-linking experiments will be performed with purified t-PA variants and supernatants of PC9 and PC9ER cells to investigate the interactions between t-PA and proteins secreted by NSCLC cells. Neuer Aspekte
Quantitative secretome analysis of NSCLC cell lines and interaction studies between t-PA and secreted proteins by chemical cross-linking and MS. Referenzen [1] Ferley J, Autier P, Heanue M, Colombet M, Boyle P, Estimates of cancer incidence and mortality in Europe in 2006, Ann.Oncol., 18, 581-592 (2007); [2] Janssen-Heijnen M L,Coebergh J W, The changing epidemiology of lung cancer in Europe, Lung Cancer, 41, 245258 (2003); [3] Ong S E, Mann M, Mass spectrometry-based proteomics turns quantitative, Nature Chem. Biol., 1, 252-262 (2005); [4] Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B, Quantitative mass spectrometry in proteomics: a critical review, Anal. Bioanal. Chem., 389, 1017-1031 (2007)
Human bone marrow stromal cell-derived osteoblast matrix vesicle proteome and functions are regulated by sulfated glycosaminoglycan derivatives Schmidt, Johannes (1); Kliemt, Stefanie (1,2); Preissler, Carolin (3); Möller, Stephanie (4); von Bergen, Martin (1,5); Hempel, Ute (3); Kalkhof, Stefan (1) 1: Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Germany; 2: B CUBE Center for Molecular Bioengineering, TU Dresden, Germany; 3: Institute of Physiological Chemistry, TU Dresden, Germany; 4: Biomaterials Department, INNOVENT e.V., Jena, Germany; 5: Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark Stichworte: Osteoblasts, matrix vesicles, extracellular matrix, glycosaminoglycans Einleitung The bone formation and the remodeling is a highly complex process that involves among others osteoblasts. These cells synthesize the extracellular matrix (ECM) and initiate its mineralization. Osteoblast-released matrix vesicles (MV) where shown to have an outstanding role in the initiation of the ECM mineralization and additionally possess proteins with different functions [1]. It is known that mesenchymal stromal cells and osteoblasts respond to the mechanical and chemical properties of their microenvironment [2]. E.g., sulfated glycosaminoglycan (GAG) derivatives where shown to promote osteoblast functions like the ECM synthesis and mineralization [3]. To delineate the molecular effects of different GAGs on the MV biogenesis, release and composition, we isolated MV from SILAC-labeled osteoblasts and quantified their respective protein composition using nano-HPLC/nano-ESI-MS/MS. Experimenteller Teil For a SILAC approach Human bone marrow stromal cells (hBMSCs) were labeled for 34 days. 7,000 labeled hBMSCs/cm² were cultured on Tissue culture polystyrene and treated with different soluble GAGs: non-sulfated hyaluronan (HA), singly-sulfated hyaluronan (sHA1), or chondroitin sulfate (sCS1). hBMSCs were differentiated into osteoblasts in osteogenic medium. The medium was changed every 3-4 days and collected until day 22. MVs were enriched by a centrifugation procedure with a final ultracentrifugation at 100,000xg for 60 min. MV and cell samples were lysed and separated by 1D-SDS-PAGE. Measurements were conducted by LTQ-Orbitrap XL ETD after nUPLC peptide separation using a 1-40 % non-linear gradient of acetonitrile. To confirm the proteomics results biochemical parameters were analyzed using immunofluorescence microscopy, Westernblot, zymography, and ELISA. Ergebnisse Initial results showed a strong promoting effect of sHA1 on the osteoblast activity by increasing the activity of the tissue non-specific phosphatase (TNAP) – an early marker of osteogenesis - as well as the calcium and phosphate deposition. Previous studies revealed only a slight impact of sulfated hyaluronan on cellular proteome of osteoblasts [3] suggesting other processes being involved. Indeed, proteomic analyses of MV fraction revealed 385 proteins of those 273 were quantified. The unusable high proportion of about 15 % regulated proteins after sHA1 treatment (FC ± 0.5, p‑value < 0.05) indicates an altered MV composition to hold a key role in the promoted ECM mineralization. The purity of the MV protein fraction was evaluated based on three criteria: (i) 21 of the major 25 extracellular vesicle (EV) proteins were present, (ii) a majority of 73 % of the identified proteins were either shown or predicted to be part of EVs and (iii) only a minority of 23 % of the identified proteins include a signal peptide to be secreted by classical mechanisms. A functional clustering of regulated proteins after sHA1 treatment indicates a promoted growth factor bioavailability by LTBP2 and fibrillin as well as activation by thrombospondins. Additionally, the cell-matrixinteraction and a proper ECM organization seem to be increased by different proteins, e.g. fibronectin and periostin. The other tested GAGs do not alter the MV composition in the same extents. sCS1 seem to have an additional influence on the cell adhesion whereas HA do not show any additional bone formation-specific influence. Taken together sHA1 seem to promote the ECM mineralization in a specific way. This seems to be relied to a large extent on an altered MV composition, but the underlying mechanisms still need to be revealed. Neuer Aspekte
We showed by quantitative proteomic and functional assays that osteoblasts MV composition and activity can be modulated by sulfated GAGs. Referenzen [1] Wuthier, R. E., and Lipscomb, G. F. (2011) Matrix vesicles: structure, composition, formation and function in calcification. Front Biosci-Landmrk 16, 2812; [2] Hempel, U. et al. (2014) Artificial extracellular matrices with oversulfated glycosaminoglycan derivatives promote the differentiation of osteoblast-precursor cells and premature osteoblasts. Biomed Res Int 2014, 938368; [3] Kliemt, S. et al. Sulfated hyaluronan containing collagen matrices enhance cell-matrix-interaction, endocytosis, and osteogenic differentiation of human mesenchymal stromal cells. J Proteome Res 12, 378-389
Monitoring PPARγ induced changes in glycolysis by selected reaction monitoring mass spectrometry Hentschel, Andreas; Coman, Cristina; Ahrends, Robert Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V. Stichworte: Adipocytes, selected reaction monitoring (SRM), SIS peptides, absolute quantification, Proteomics Einleitung The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear fatty acid receptor that has been shown to be the ultimate regulator of fat cell differentiation [1,2]. The realization that a fatty acid sensor such as PPARγ might also be an important regulator of glucose metabolism arose from the discovery that the insulin-sensitizing thiazolidinediones (TZDs) such as rosiglitazone are potent agonists for PPARγ [3] leading to an upregulation of the glucose transporter GLUT4. Because of this regulatory function in glucose metabolism, it is of great interest to monitor the relative and absolute changes in glycolysis upon PPARγ activation. Since PPARγ is the metabolic master regulator of many processes, we used this model system investigate cellular metabolic remodeling. Experimenteller Teil To screen and identify the glycolytic and other main regulated pathways upon PPARγ activation, we first set up a label free quantitative global proteomics approach by using a high resolution mass spectrometry system (QExactive Plus). By applying STRING 9.1 and Cytoscape 3.2.0 we visualized the regulated proteins, pathway and network dynamics. To further investigate the regulatory effects of PPARγ on different pathways, we performed a titration of rosiglitazone and analyzed the samples with a targeted proteomics approach by using selected reaction monitoring (SRM). This allowed for deeper investigation of each pathway by having a higher sensitivity, absolute amounts and wider dynamic range. Ergebnisse Here we established a quantitative proteomics approach to analyze and quantify proteins of the glycolytic and other metabolic pathways by using a global label free and a targeted proteomics setup. Our results show in general the regulation of 719 proteins quantified with at least 2 peptides. 23 enzymes and their isoforms which are involved in the glycolytic pathway could be identified and absolute quantified using stable isotope labeled standard peptides (SIS peptides). The rosiglitazone titration experiment revealed that certain key players such as the ADP-dependent Glucokinase, Hexokinase (initial steps) and Phospho-fructo-kinase (rate limiting step) display a significant upregulation during the PPARγ activation while the level of regulation and abundance is reverse correlated with their enzymatic activity. A first closer look at those regulated proteins revealed them as potential downstream targets of PPARγ. Moreover there is an evidence for a metabolic shift from the glycolysis pathway to other metabolic pathways which is not controlled by PPARγ. With this set of methods and system we want to further investigate the networks of lipid metabolism controlled by PPARγ activation to reveal the detailed dynamics of protein and pathway regulation and possible metabolic shifts. Neuer Aspekte Absolute quantification of abundance changes in the glycolytic pathway upon PPARγ activation using SIS peptides and SRM. Referenzen [1] Rosen, E.D., et al., PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell, 1999. 4(4): p. 611-7.; [2] Ahrends, R., et al., Controlling low rates of cell differentiation through noise and ultrahigh feedback. Science, 2014. 344(6190): p. 1384-9.; [3] Lehmann, J.M., et al., An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferatoractivated receptor gamma (PPAR gamma). J Biol Chem, 1995. 270(22): p. 12953-6.
Proteomic study of the impact of Magnesium implants on osteoblasts Omidi, Maryam (1); Burmester, Anna (2); Kiani, Parnian (1); Kwiatkowski, Marcel (1); Luthringer, Berengere (2); Willumeit, Regine (2); Schlüter, Hartmut (1) 1: Department of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2: Helmholtz-Zentrum Geesthacht; Institute of Materials Research; Structural Research on Macromolecules; Geesthacht, Germany Stichworte: Magnesium, Implant, Proteomic, mass spectrometry Einleitung Magnesium implants for bone healing offer the opportunity getting degraded by corrosion and thus avoiding a second surgery for removing them, because the implants in the best case will have been disappeared after bone healing will have been finished (1 and 2). By the corrosion process Mg2+ ions are formed which will increase the Mg2+ concentration significantly above physiological levels (1). Thus, the question arise, how these elevated Mg 2+ levels affect bone cells. Therefore, we studied the effect of elevated Mg2+ levels on differentiated and proliferated osteoblasts by proteomics. Experimenteller Teil In this study, we investigated the proteomes of cultured proliferating and differentiating osteoblast, in absence (control) and presence of 5mM MgCl2, and Mg-alloy extracts. Cell pellets were lysed; proteins of these cells were extracted, reduced, alkylated and finally incubated with trypsin. Tryptic peptides were subjected to a nano-UPLC-column coupled to a hybrid orbitrap system (Orbitrap-Fusion, Thermo Fisher scientific). Data analysis was performed with Proteome Discoverer (Thermo Fisher scientific) and a first data interpretation for a rough preliminary comparison of relative protein abundance done by using spectral counting. Ergebnisse 370000 spectra were recorded in total yielding approximately 4700 identified proteins including more than 10000 peptides. The levels of hundreds of proteins increased respectively decreased in response to elevated concentrations of Mg2+. The level of Neuroblast differentiation-associated protein (AHNAK), a 700-kDa scaffold protein, was massively associated with the increase of Mg2+ concentrations and thus attracted attention. AHNAK is involved in regulation of voltage-gated calcium channel activity, thus obviously being an important protein in regulating the physiological actions of osteoblasts. The level of AHNAK is decreased in differentiating osteoblasts in presence of elevated Mg2+ levels in comparison with differentiating osteoblast in absence of Mg2+. In contrast, the AHNAK abundance in proliferating osteoblasts is increased in presence of elevated Mg2+ compared with proliferating osteoblast in absence of Mg2+. In summary, the investigation of the response of osteoblasts towards elevated Mg2+ with proteomics tools gave deep insights in the Mg2+ induced changes in the protein composition of the osteoblasts. Linking these changes to molecular physiology of the osteoblasts the overall response of osteoblasts to elevated Mg2+ seems to be beneficial for bone healing. Acknowledgements Financial support by the Helmholtz Virtual Institute MetBioMat is gratefully acknowledged. Neuer Aspekte Comprehensive proteomics study of the effect of magnesium implants on bone cells Referenzen
[1] Anna Burmester, Bérengère Luthringer, Regine Willumeit, Frank Feyerabend, Comparison of the reaction of bone-derived cells to enhanced MgCl2-salt concentrations, Biomatter, 4:1, e967616, DOI: 10.4161/21592527.2014.967616.; [2] Witte F, Kaese V, Haferkamp H, Switzer E, Meyer-Lindenberg A, Wirth CJ, Windhagen H., In vivo corrosion of four magnesium alloys and the associated bone response, 2005, Biomaterials 26, 3557–3563.
Uncontrolled modification of redox-active cysteines in proteins and peptides Bayer, Malte; König, Simone Core Unit Proteomics, Interdisciplinary Center for Clinical Research, University of Münster Stichworte: artifactual protein modifications, redox-active cysteines Einleitung Modifications of proteins caused by sample handling interfere with the detection of posttranslational modifications. Phosphorylation is a case in point, as it may easily be confused with artifactual sulfonation (Δ 9.5 mDa). Moreover, both real and artificial modifications are typically of low abundance increasing the analytical difficulties further. It has been, e.g., shown that artifactual sulfation of hydroxyamino acids can occur during silver staining [1]. S-sulfonation of cysteine has also been described for transthyretin when incubated at 37 °C under alkaline conditions for one week [3]. Here we present evidence that S‑sulfonation alongside other modifications of redox-active cysteines can occur as a result of standard proteomic work-up procedures, such as tryptic digest and SDS-PAGE. Experimenteller Teil Commercial standard proteins (Sigma-Aldrich) and synthetic peptides (Thermo Scientific) were subjected to proteomic routine procedures. Proteins were purified by SDS-PAGE using 12 % SDS‑polyacrylamide gels and stained by blue silver staining [2]. Gel pieces were destained and digested with trypsin (Serva) in NH 4HCO3‑solution (50 mM, pH 8.7, Fluka) at 37 °C overnight. Alternatively, the proteins were directly trypsinized without prior purification steps. Synthetic peptides were heated to 95 °C for 5 min (pH 6.8) and incubated overnight at 37 °C in NH4HCO3‑solution (50 mM, pH 8.8) with 0.05 % (NH4)2S2O8 (w/v, USB Corporation) to simulate an SDS‑PAGE. LC-MS/MS was performed using C18 nanoUPLC-tandemMS (nanoAcquity, Q-TOF Premier, Waters Corp.). Ergebnisse Peptides of interest (containing “redox-active” cysteines) were selected based on the close proximity of a basic amino acid to cysteine. The addition of nominal 80 Da was detected after SDS-PAGE or tryptic digest in every examined sequence. Additional cysteine modifications were also found (+76 Da, +64 Da, +48 Da, +32 Da, -34 Da). The formation of dehydroalanine and thiocysteine (-34 Da, +32 Da) has been suggested before [3]. Disulfide formation with β‑mercaptoethanol (+76 Da) has also been discovered by other authors [4]. This modification must be expected in commercial products and synthetic peptides even when the use of β‑mercaptoethanol is not explicitly stated as it is a common agent in protein purification procedures. The addition of +48 Da might either be attributed to oxidation to cysteic acid or to S-sulfenation whereas the +64 Da modification most likely represents S-sulfination. Only when adding 10 mM DTT to every step of the work-up including the sample buffer, modifications of +76/80 Da were eliminated. Neuer Aspekte Proteomic procedures cause artifactual modifications of redox-active cysteines Referenzen [1] Gharib, M; Mol. Cell Proteomics; 2009; 8(3); 506-518; [2] Dyballa N.; Laborjournal; 2008; 4; 44-45; [3] Nakanishi, T.; Biochim. Biophys. Acta; 2004; 1698; 45-53; [4] Begg, G. E.; J. Biomol. Tech.; 1999; 10(1); 17-20
Low abundant N-linked glycosylation in wild-type hen egg white lysozyme is localized at non-consensus sequons Asperger, Arndt (1); Marx, Kristina (1); Albers, Christian (1); Molin, Laura (2); Pinato, Odra (2) 1: Bruker Daltonik GmbH, Bremen, Deutschland; 2: Chelab Silliker, Resana, Italy Stichworte: Hen egg white lysozyme, non-consensus N-glycosylation sites, glycan heterogeneity, LC-MS/MS, GlycoQuest bioinformatics Einleitung Hen egg white lysozyme (HEL) is one of the most extensively studied glycoside hydrolase enzymes, and was the first enzyme, for which a crystal structure had been published. Although wild-type HEL is lacking the consensus sequence motif NX(S/T), in 1995 Trudel et al.1 proposed the existence of a low abundant N-glycosylated form of HEL. However, the identity of active glycosylation sites in HEL remained a matter of speculation. For the first time since Trudel´s initial work, we report here a comprehensive characterization by means of mass spectrometry of the proposed low abundant N-glycosylation in wild-type HEL aiming for the unambiguous identification of active glycosylation sites and for profiling the compositional heterogeneity of the modifying N-glycans. Experimenteller Teil HEL was digested in solution using trypsin as a cleaving enzyme. From the resulting digest, the N-glycopeptides were enriched by means of ZIC-HILIC. Subsequently, the enriched N-glycopeptides were enzymatically deglycosylated by 18 O/PNGase F and various endoglycosidases (F1, F2, F3, H). Both the intact and deglycosylated N-glycopeptides were analyzed by LC-MS/MS employing multiple mass spectrometric techniques (MALDI-TOF/TOF, ESI-iontrap using either CID or ETD for fragmentation). Data analysis was performed using Bruker´s ProteinScape 3.1 bioinformatics platform featuring a novel data interpretation workflow, consisting of MS/MS spectra classification and glycan database searching, enabling the efficient automatized detection and characterization of N-glycopeptides in LC-MS/MS datasets obtained from glycoprotein digests. Ergebnisse LC-MS/MS data obtained from the intact N-glycopeptides enriched from HEL trypsin digest enabled the identification of the asparagine-rich N-glycopeptide [34-45] FESNFNTQATNR containing three Asn residues (N37, N39 and N44) as potential glycosylation sites. The automated detection of this N-glycopeptide was facilitated by the novel GlycoQuest data interpretation workflow featured in the ProteinScape 3.1 software used here. Furthermore, the intact N-glycopeptide data were utilized to profile the heterogeneity of the N-glycans modifying HEL. The novel GlycoQuest search engine was employed to identify the composition of the glycan moieties. Overall, the heterogeneity profile of N-glycans in HEL comprised at least 26 different glycan compositions. Additional deglycosylation experiments were required to achieve the unambiguous identification of the active sites of N-glycosylation in HEL. LC-MS/MS results obtained after deglycosylation with 18O/PNGaseF and endoglycosidase F/H, respectively, provided clear evidence for Asn residues N44 and N39 as active glycosylation sites in HEL. These sites are part of the non-consensus sequons NXN and NXQ, respectively, which have only been reported in very few cases before for glycoproteins of eukaryotic origin. Neuer Aspekte Identification of previously unknown N-glycosylation sites in HEL; profiling the N-glycan heterogeneity; novel bioinformatics workflow for glycopeptide analysis Referenzen [1] J. Trudel, A. Asselin, Biochem. Cell Biol. 73: 307-309 (1995)
A closer look at the quality of microwave digests Jüngst, Vera; Bäumlisberger, Marion; Bahr, Ute; Karas, Michael Goethe Universität Frankfurt, Deutschland Stichworte: Microwave protein digestion, rel. quantification, MALDI-MS Einleitung Protein digestion and digestion time is still a bottleneck in proteolytic analyses. For accelerating digestion microwaveassisted protein digestion has been reported as a promising technique with digestion times of even < 30 min. Preliminary results presented on the 2014 DGMS meeting showed that there are no distinct differences between digests in microwave system compared to a water bath under comparable temperature and time conditions. These data were in accordance with published results. In most cases the sequence coverage or the number of identified peptides was used as measure for comparison. [1] Some authors used the quantity of single marker peptides or the absence of the intact protein as indicator for the completeness of the digest. [2] Experimenteller Teil For evaluation of the microwave digests we used a CEM Discover System (CEM, Matthews, USA) with a maximum power output of 300 W and a frequency of 2.45GHz and compared it with a thermoshaker. Apo-transferrin and myoglobin were taken as standard proteins and digested with the commonly used highly specific serine protease trypsin (E:P = 50). As an internal standard for relative quantification [Glu 1]-fibrinopeptide (Glu-Fib) was used and mixed with α-cyano-4-hydroxycinnamic acid (CHCA). The experiments covered a digestion time range of 30 min to 18h and were performed with and without prior reduction and alkylation. For optimization of the digest results the anionic detergent sodium deoxycholate was added to some samples.
Ergebnisse When comparing microwave and thermoshaker at equal time points and temperatures, again no superiority of the microwave could be detected with respect to sequence coverage. While the sequence coverage often reaches a maximum after short times and stays constant over a long period it is expected that the total peptide number should increase over a longer time interval until the protein is completely digested. For a detailed comparison of microwave and shaker digests a relative quantification of digestion peptides was performed using Glu-Fib as internal standard. Evaluation of all peptides of the protein used for Mascot search without missed cleavages and modifications over the time shows that the intensity and accordingly the quantity of the peptide not necessarily increases with time. Different peptides show different time profiles. The shape of these profiles is comparable between microwave and shaker with a slight superiority of the thermoshaker. The results show, that it is not meaningful to take a random peptide as a quantitative marker over time and that the sequence coverage is not a reliable marker for digestion efficiency. The addition of sodium deoxycholate prior the digestion increases the sequence coverage - especially for non-reduced and alkylated samples. The quantitative evaluation of samples with addition of sodium deoxycholate shows a higher amount of some peptides. Neuer Aspekte Quantification of many peptides to compare microwave and shaker digests. Referenzen [1] H.-F. Juan, S.-C. Chang, H.-C. Huang, und S.-T. Chen, „A new application of microwave technology to proteomics“, PROTEOMICS, Bd. 5, Nr. 4, S. 840–842, 2005.;
[2] J. L. Proc, M. A. Kuzyk, D. B. Hardie, J. Yang, D. S. Smith, A. M. Jackson, C. E. Parker, und C. H. Borchers, „A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Tryp
New Workflows for Identification and Profiling of Disulfide Bonds in Biopharmaceuticals Wiesner, Jan; Kozicki, Antje; Resemann, Anja; Paape, Rainer; Vorwerg, Lars; Marx, Kristina; Kiehne, Andrea; Hartmer, Ralf; Baessmann, Carsten; Suckau, Detlev; Jabs, Wolfgang; Centnar, Zoltan Bruker Daltonik GmbH, Deutschland Stichworte: Disulfide bond, ETD, ion trap, protein identification, biopharmaceutical Einleitung The spatial structures of biologics are crucial for their safety and efficacy and fundamentally determined by disulfide bonds (DSBs). We evaluated alternative approaches to the classical comparison of reduced and non-reduced digests using ESI LC-MS³ and MALDI LC-MS³ and non-reduced protein digests.. These workflows were applied to various model proteins (α-lactalbumin, RNAse, adalimumab) Experimenteller Teil Digests of were performed with a mixture of LysC and trypsin to obtain a good yield. Free cysteins were blocked with N-ethylmaleimide to avoid disulfide scrambling . The samples were separated using nano-LC followed by MS analysis. An ion-trap equipped with a CaptiveSpray ionization source was used in the ETD workflow, and a MALDITOF/TOF instrument was used in the ISD workflow. Software was developed to identify characteristic fragmentation patterns of disulfide-linked peptides in MS2 spectra and generate target lists for the MS3 step. Ergebnisse To improve ETD efficiency, sheath gas was acetonitrile-enriched : the average charge state distribution (CSD) of the analyzed DSB-peptides was shifted by 30% towards the higher values, and their average intensity was doubled. In this approach, we utilized a new targeted MS3 experiment to profile DSB-peptides by applying ETD in MS2 followed by targeted CID in MS3. we acquired retention time dependent full scan MS3 spectra in the IT, enabling simultaneous detection of many peptide fragments. .With this targeted workflow, 7 of 8 expected adalimumab DSBpeptides were identified. MALDI-ISD was used to partially reduce LC separated DSB-peptides. For the identification of DSB-peptides, MS spectra were screened for triplets of the DSB-peptide and their two characteristic ISD fragments. All peaks were subjected to LC-MALDI-MS/MS analysis. For adalimumab, all DSB-peptides with one disulfide bond were identified with this method. Neuer Aspekte Protein disulfide bond analysis was performed using either an ETD-MRM workflow with an ion-trap or a new MALDI-ISD workflow
Exploring the potential of the last generation UHR-Q-TOF for rapid generation of accurate information on proteoforms distribution and relative abundancy Jabs, Wolfgang; Pengelley, Stuart; Albers, Christian; P.O Schmit, P.O Schmit; Tellström, Verena Bruker Daltonik GmbH, Deutschland Stichworte: Proteoform, top-down, QTOF, intact proteins Einleitung All along their life cycle, proteins undergo various transformations that can alter their functions while keeping a good part of their primary sequence intact. These multiplications of PTM patterns, alternative splicing forms or products of proteolitic processing cannot be simply resolved with a bottom-up approach, as very few peptides are specific from the given proteoform. However, the information relative to the distribution of different proteoforms is encoded in their intact masse. In this study we were willing to evaluate how the latest generation UHRQ-Tof capabilities of delivering resolution, mass accuracy and isotopic fidelity even in highly complex mixtures separated with Fast LC could help to quickly resolve proteoforms in complex mixtures, in order to map their distribution and relative abundancies.
Experimenteller Teil Undigested protein mixtures of E.Coli ,Yeast and non-depleted plasma were separated on a Phenomenex Aeris Widepore, 3.6 µ, C4, 150x2.1 mm column. LC was performed on a Dionex RSLC system coupled to an impact II (Bruker) or maXis II UHR-Q-Tof (Bruker) , operating in MS, Auto MS/MS or targeted MS/MS acquisitions. Mixtures of yeast intact protein (Promega) spiked with various ratios of UPS2 Proteomics Dynamic Range Standard Set (Sigma) were separated on a 250 mmX100 µm monolithic pepswift PS-DVB column after preconcentration on a monolithic pepswift 200µm x 5mm trap. LC was performed on an Ultimate nano-RSLC system (Thermo) coupled to an impact II (Bruker) via a CaptiveSpray nanoBooster ion source (Bruker), and operated in MS and auto MS/MS modes. Ergebnisse Using High-Quality threshold protein detection, we could easily detect more than 800 proteoforms (from doubly charges peptides up to 35Kda proteins) out of the E.Coli mixture, and more than 1500 proteoforms out of 50µg of the Yeast mixture, using a 15 minute gradient (20 minutes method). The separation of yeast over a 35 minutes gradient (45 minutes method) enabled to distinguish more than 1000 proteoforms from a 1µl injection of non-depleted plasma. The spectral quality observed for single compounds was preserved while measuring these highly complex mixtures : the average mass error for the monoisotopic peak of the RS 19 protein (10,2 Kda) over 4 injections was 0,07 ± 0,2 ppm. The isotopic fidelity (express as shift of the theoretical abundance, relative to the most abundant isotope) was always better that 2%, and the resolution exceeded 50 000. An Auto-LC MS/MS run enabled the identification of RS 19 using CID from several charge states. In the absence of on-the flight deconvolution, automated LC-MS/MS enabled to fragment the high abundance low MW (2000 Einträge). Das Verfahren basierte auf der Suche nach Suspects, die über die exakte Masse ermittelt und anschließend über Isotopenmuster und Fragment-Ionen bestätigt wurden. Für das Non-Target Screening wurden online Datenbanken wie STOFF-IDENT oder das Biotransformationsvorhersagemodell EAWAG-BBD herangezogen. Ergebnisse Mit Hilfe des Suspect Screening konnten 93 Verbindungen ermittelt werden. Davon wurden 60 Verbindungen erstmalig im Projektgebiet detektiert: 32 Arzneistoffe, neun Arzneistoff-Metabolite, zwei Pestizide, drei PestizidMetabolite und 14 sonstige Spurenstoffe. 58 Verbindungen wurden insgesamt quantifiziert, 21 Verbindungen dabei erstmalig in dem Projektgebiet. Im See hatten 46 Verbindungen eine Konzentration größer der Bestimmungsgrenze, sechs sogar mit einer Konzentration größer 1,0 µg/L. Im Rohwasserbrunnen wurden noch 31 Verbindungen detektiert. Hier lagen 11 Verbindungen vor, die den allgemeinen Vorsorgewert des Umweltbundesamt (0,1 µg/L) überstiegen. Mit den Quantifizierungsdaten wurde das Transportverhalten in der Transekte untersucht. Die Messstellen haben unterschiedliche Teufen. Dadurch können innerhalb der Transekte die verschiedenen Redoxbereiche abgedeckt werden, um mikrobielle und chemische Prozesse zu charakterisieren. Einige der mit dem Suspect Screening neu detektierten Verbindungen zeigten sich während der Uferfiltration persistent, was im Hinblick auf die Gefährdung der Trinkwasserressourcen relevant ist. So konnte für die Blutdrucksenker Candesartan und Olmesartan ein redoxabhängiges Abbauverhalten nachgewiesen werden. Diese verhielten sich im Aeroben persistent, während im Anaeroben ein Abbau stattfand. Das Antiepileptikum Gabapentin und das Transformationsprodukt der Sartane, die Valsartansäure, verhielten sich dagegen unabhängig vom Redoxmilieu persistent. Während Verbindungen wie das Analgetikum Tramadol und das Diuretikum Hydrochlorothiazid in allen Redoxmilieus Abbau zeigten.
Über das Non-Target Screening konnten des Weiteren drei potentielle Transformationsprodukte mit dem EAWAGBBD vorhergesagt werden. Diese stellen vermutlich Derivate des Phenazons (Acetylierung), des Propyphenazons (Hydroxylierung) und des PDPs (Formylierung des Abbauprodukts von Propyphenazon) dar. Eine Verifzierung dieser Verbindungen konnte aufgrund fehlender Referenzstandards nicht vorgenommen werden. Ein anderes Transformationsprodukt, das Gabapentin Lactam, wurde ebenfalls über das Screening identifiziert. Gabapentin Lactam resultiert aus Gabapentin durch Wasserabspaltung und intramolekularem Ringschluss und konnte sogar mittels eines Referenzstandards verifiziert werden. Neuer Aspekte Spurenstoffcharakterisierung einer Uferfiltrationstransekte mittels UHPL-HRMS Screening und Quantifizierung zur Bewertung des Berliner Grundwassers. Referenzen [1] S. Grünheid, G. Amy, and M. Jekel, “Removal of bulk dissolved organic carbon (DOC) and trace organic compounds by bank filtration and artificial recharge.,” Water Res., vol. 39, no. 14, pp. 3219–28, Sep. 2005.; [2] F. Wode, P. van Baar, U. Dünnbier, F. Hecht, T. Taute, M. Jekel, and T. Reemtsma, “Search for over 2000 current and legacy micropollutants on a wastewater infiltration site with a UPLC-high resolution MS target screening method.,” Water Res., vol. 69C, pp; [3] F. Wode, C. Reilich, P. van Baar, U. Dünnbier, M. Jekel, and T. Reemtsma, “Multiresidue analytical method for the simultaneous determination of 72 micropollutants in aqueous samples with ultra high performance liquid chromatography-high resolution mass sp
A DESI MS based screening method for phthalates in consumer goods Schulz, Sabine (1); Wagner, Sebastian (1); Gerbig, Stefanie (1); Bohn, Dieter (2); Sielaff, Detlef (3); Wächter, Herbert (4); Spengler, Bernhard (1) 1: Justus-Liebig-Universität, Deutschland; 2: Landesbetrieb Hessisches Landeslabor, Deutschland; 3: Landesuntersuchungsamt Rheinland-Pfalz, Deutschland; 4: Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Deutschland Stichworte: DESI, Phthalates, toys, screening, quantitation Einleitung Phthalates are used as plasticizers in many everyday items, but they are also recognized as hormone disruptors being especially harmful during childhood [1]. The European Union therefore restricted their use in children’s toys and certain food packaging [2].Due to the ever-increasing number of polymer-containing consumer goods, rapid screening methods are needed to ensure and improve consumer safety in the future. In this study we evaluated the performance of desorption electrospray ionization (DESI) mass spectrometry (MS) for rapid quantitative screening of phthalates in consumer goods. Experimenteller Teil Analysis of matrix-matched reference materials and authentic samples was carried out using a custom-built DESI source attached to an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany). Reference materials were prepared by mixing PVC powder and plasticizer, followed by heating of the gel for 20 min to 200°C in a mold. Authentic samples were analyzed without further sample treatment. Samples and reference materials were fixed to a motorized 2D stage and measured in an automated fashion, with the MS operating in positive ion mode. Additional experiments were performed with the DESI source being attached to a portable MS Mini 11 [3] (Purdue University, USA). Determined calibration curves show the TIC-normalized sum of phthalate ion signals against true concentrations. Ergebnisse In this study a DESI MS method for rapid identification and quantification of phthalates in consumer goods was developed. DESI allowed for direct surface sampling of the consumer goods under atmospheric conditions with minimal sample preparation, while the high-performance mass spectrometer provided for high sensitivity and reliable identification via accurate mass measurements, high mass resolving power and MS/MS capabilities. External calibration curves for six banned phthalates (DBP, BBP, DEHP, DNOP, DINP and DIDP) were obtained from matrixmatched reference materials. Coefficients of determination were greater than 0.985, LOQs ranged from 0.02 to 2.26 %w (legislative limit: 0.1%w) and the relative standard deviation of calibration-curve slopes was less than 7.8 % for intraday and 11.4 % (DEHP) for interday comparisons. Measurement of a calibration curve (12 points) was achieved within three minutes. The phthalate contents of eleven authentic samples were determined in a proof-of-concept approach using DESI MS, and results were compared to those from confirmatory methods. Sample preparation for authentic samples involved the attachment of a small piece to a sample holder and less than a minute for analysis. The phthalate contents were correctly assigned for the majority of samples within a range of -20 to +10 % relative deviation to the results obtained with confirmatory methods. Compared to confirmatory methods such as gas and liquid chromatography coupled with spectroscopic and spectrometric detection, the DESI MS method constitutes a major reduction in total analysis time, due to shorter measurement times and omitted sample preparation. Given further optimization and automation, DESI MS might become a useful tool for rapid and accurate phthalate screening in the future. Currently a field-portable instrumental setup is tested in on-site screening experiments. Neuer Aspekte Quantitative DESI MS based method for screening of phthalates in consumer goods. Referenzen
[1] S.H. Swan, F. Liu, M. Hines, R.L. Kruse, C. Wang, J.B. Redmon, A. Sparks, and B. Weiss, Prenatal phthalate exposure and reduced masculine play in boys. International Journal of Andrology 33 (2010) 259-267.; [2] DIRECTIVE 2005/84/EC of the European Parliament and the Council, 2005; [3] Gao L, Sugiarto A, Harper JD, Cooks RG, & Ouyang Z. Design and characterization of a multisource hand-held tandem mass spectrometer. Analytical chemistry 2008; 80: 7198-7205.
Identifizierung von intrazellulären Cisplatin-Protein Addukten mithilfe des fluoreszierenden CisplatinAnalogon CFDA-Cisplatin Kotz, Sandra (1); Kullmann, Maximilian (2); Kalayda, Anya (2); Jaehde, Ulrich (2); Metzger, Sabine (1) 1: Universität zu Köln, Deutschland; 2: Universität Bonn, Deutschland Stichworte: Cisplatin, CFDA-Cisplatin, 2D-Gelelektrophorese, CFDA-Cisplatin Addukte Einleitung Cisplatin ist ein breit eingesetzter Wirkstoff in der Krebstherapie. Derzeit erfolgt eine Behandlung mit Cisplatin bei Plattenepithelkarzinome an Kopf und Hals, Bronchial-, Darm-, Harnblasen-, Hoden- und Ovarialkarzinom [1-3]. Seine Wirksamkeit ist aber durch toxische Nebenwirkungen sowie durch eine Resistenz von Tumorzellen gegenüber Cisplatin begrenzt. Die zytotoxische Wirkung von Cisplatin beruht auf der Platinierung der DNA [4], aber nur ein geringer Anteil des zellulären Cisplatins gelangt zum Zielort [5]. Es wird angenommen, dass Cisplatin eine höhere Affinität zu schwefelhaltigen Aminosäuren wie Cystein und Methionin hat [6]. Im Inneren einer Tumorzelle gibt es für Cisplatin eine Reihe von möglichen Bindungspartnern. Daher kann für Cisplatin die Wechselwirkung mit Proteinen ein wichtiger Faktor für dessen intrazelluläre Verteilung, Elimination und Zytotoxizität sein [7]. Experimenteller Teil Zur Identifizierung und Auftrennung solcher intrazellulären Cisplatin-Protein Addukte wurde mithilfe des fluoreszierenden Cisplatin-Analogon CFDA-Cisplatin [8] eine 2D-Gelelektrophorese etabliert. Dieses fluoreszierende Cisplatin-Analogon ermöglichte die Detektion bzw. Unterscheidung von intrazellulären Cisplatin-Protein Addukten. Um die Komplexität und Auflösung von sauren, neutralen sowie basischen Proteinen zu erhöhen, erfolgte die Auftrennung mithilfe der 2D-Gelelektrophorese in teilweise überlappende immobilisierte pH-Gradienten (pH 4-7 und 6-10). Die detektierten CFDA-Cisplatin-Protein Addukte wurden mittels ESI-MS/MS identifiziert. Ergebnisse Mit dieser Strategie konnte der Elongation factor 1-alpha 1, die D-3-phosphoglycerate dehydrogenase und einige Protein-Disulfid-Isomerasen identifiziert werden. Neuer Aspekte Dies sind die ersten Ergebnisse zur Detektion von intrazellulären Cisplatin-Protein Addukten. Referenzen [1] [1] Galanski et al. 2006; [2] Lebwohl et al. 1998; [3] Prestayko et al. 1979; [4] Jamieson et al. 1999; [5] Cepeda et al. 2007;; [2] [6] Kasherman et al. 2009; [7] Galluzzi et al. 2012; [8] Molenaar et al. 2000
Veränderungen des organischen Bodenkohlenstoffs bei Waldbränden Hofmann, Diana (1); Steffen, Bernhard (1); Eckhardt, Kai-Uwe (2); Leinweber, Peter (2) 1: Forschungszentrum Jülich, Deutschland; 2: Bodenkunde der Universität Rostock Stichworte: ESI-FTICR-MS, Kendrick-Diagramm, Neubildung, stickstofforganische Verbindungen, Van KrevelenDiagramm Einleitung Thermisch entstandene, hocharomatische Kohlenstoffverbindungen, sogenannter „Black Carbon“ wird aufgrund seiner Klimarelevanz seit Jahren intensiv untersucht. Aufgrund seiner schweren Abbaubarkeit reichert er sich in Böden an – Anteile am Gesamt-C variieren zwischen 1-6 % im europäischen Waldboden und bis zu 60 % in den Schwarzerden - und stellt somit eine Senke im globalen Kohlenstoffkreislauf dar. Unbekannt ist, inwieweit Anteile des Black Carbon wasserlöslich sind und den Pool an mobilen gelösten organischen Substanzen qualitativ und quantitativ beeinflussen. In der Präsentation wird dargestellt, wie sich der (wasserlösliche Teil des) organischen Bodenkohlenstoffs unter Hitzeeinwirkung als Simulation von Waldbränden verändert. Experimenteller Teil Nach der Elementaranalyse der erhitzten sowie der unbehandelten Kontrollproben aus Freiland-Lysimetern wurden Heißwasserextraktionen durchgeführt. Nach Zentrifugation und Festphasenextraktion zur simultanen Entsalzung und Aufkonzentrierung der Proben wurden diese mittels Fließinjektion in ein ESI-FTICR-MS (LTQ-FT Ultra, ThermoFisher Scientific) infundiert und als gemittelte Spektren aus 6 Scans mit jeweils 50 Transienten unter einer Auflösung von 400.000 (bei m/z = 400 Da) und mit einer Genauigkeit 0.98. For method validation, the performance of the Flowprobe system will be further compared with a cobas® 6000 analyzer series (Roche) measurement as a reference method. As a second application and an example for detection of exogenous interfering substances, contamination with the hydrophobic reagent dioctyl sulfosuccinate sodium (DONS) were analysed on irregularities on semi-finished products (coated foil) of glucose measurement tests. Taken together, our results suggest that the Flowprobe™ method is applicable for on-line extraction and detection of acetaminophen in dried blood spots on test strips as well as a contamination on coated foils. The novel Flowprobe™ system has the potential to directly analyse small molecules out of complex matrices. This can facilitate detection of interfering substances for enzyme-based glucose measurements. The main advantage of this method is a very small sample volume (< 2.5 µl) and a direct analysis from used test strips without any sample preparation. Additionally, the system can be used for detection of exogenous interfering contaminations on surfaces like a coated foil. This approach could be a rapid alternative for time-consuming in-process controls during production of test systems. Neuer Aspekte analysis of acetaminophen in dried blood on glucose test-fields and detection of DONS on foils of test-stripes as inprocess control Referenzen
[1] ZUPING TANG, XIAOGU DU, RICHARD F. LOUIE, GERALD J. KOST: Effects of Drugs on Glucose Measurements with Handheld Glucose Meters and a Portable Glucose Analyzer. In: Am J Clin Pathol 133 (2000), S. 75–86; [2] HSU, Cheng-Chih ; ELNAGGAR, Mariam S. ; PENG, Yao ; FANG, Jinshu ; SANCHEZ, Laura M. ; MASCUCH, Samantha J. ; MØLLER, Kirsten A. ; ALAZZEH, Emad K. ; PIKULA, Jiri ; QUINN, Robert A. ; ZENG, Yi ; WOLFE, Benjamin E. ; DUTTON, Rachel J. ; GERWICK, Lena ; ZHA
Improved Determination of Allergenic Fragrances in Detergents and Personal Care Products in Multiple Reaction Monitoring GC-MS/MS van’t Slot, Gordon; Tellström, Verena Bruker Daltonik GmbH, Deutschland Stichworte: allergy, MRM, database, triple quad, PCP Einleitung Many compounds added to improve the aroma of cleaning agents and personal care products (PCP) are classified as allergenic. Quantifying the risk of these to personal safety is complicated as even sub-ppb levels may invoke a serious immunogenic response in hypersensitive individuals. Therefore EU Directive No. 76/768/EC stipulates that allergens should be labelled if they exceed 0.001% (w/w) for rinse-off products and 0.01% (w/w) for stay-on products. Developers therefore require analytical techniques that can accurately and reliably screen for low level fragrance residues. GC-MS/MS in MRM mode is regarded as the gold standard for low level multiple residue analysis, particularly in matrices containing cleaning agents and PCPs. However MRM setup and method development is a complicated and time intensive process. Experimenteller Teil GC-MS/MS techniques based on compound based scanning (CBS) greatly simplify this process, combining enhanced sensitivity, reliability and robustness with rapid method development and simple sample preparation. GC: Bruker GC 456 SCION TQ; S/Sl-injector temp: 250°C; Split: 1:20; Column: BR-5ms, 30 m x 0.25 mm x 0.25 μm; Oven program: 50°C (1 min) -> 250°C (12°C/min); 3.33 min; Injection volume: 1 μL; MRM measurements with optimized transitions for all compounds (at least 2 transitions per compound); Data system: Bruker MSWS 8.0 SP2 Ergebnisse To illustrate the speed and sensitivity of employing this method for allergen screening, a GC-MS/MS was used to screen for allergenic compounds within a number of commercially available cleaning agents. CBS software automatically built the screening method while advanced GC-MS/MS hardware delivered excellent sensitivity at trace ppb levels. After a small number of initial runs to locate the retention time window for each compound, CBS software used a comprehensive MRM database to automatically select optimal scan times for the 27 allergen compounds. Calibration curves from 5 ppt to 250 ppb could be measured. The MRM traces clearly identified the presence of ten prohibited allergenic compounds screened for using the CBS method. Analysis was also performed in single quadrupole SIM mode to illustrate the improvements in selectivity in matrix samples. Usage of a triple quadrupole MS/MS offers greater s/n ratio and improved peak shapes. The majority of allergenic compounds listed in the EC regulation have been optimized in CBS scanning, which provided fast and robust method development, showing the method to be ideal for product developers and quality control (QC) laboratories. Neuer Aspekte GC-MS/MS for quantification of trace-levels of allergenic compounds in personal care products (PCP)
Direct Analysis of Degradation Products in Lithium-ion Batteries Using Low-temperature Plasma Ambient Desorption/Ionization High-Resolution Mass Spectrometry Kuhlmann, Christopher (1); Pastushkina, Inessa (1); Nowak, Sascha (2); Engelhard, Carsten (1) 1: Universität Siegen, Deutschland; 2: MEET - Münster Electrochemical Energy Technology, Deutschland Stichworte: LTP-MS, Lithium-ion battery, volatile compounds Einleitung Lithium ion batteries (LIB) are important energy storage devices due to their high energy density, relatively low selfdischarge, and low maintenance needs. LIB devices help to realize time independent and delocalized energy storage on a large scale. One limitation, however, is aging of LIBs due to degradation processes within the batteries. To improve the performance and long-term stability of these devices, it is critical to understand these degradation processes. Typically, analysis of degradation products is performed with analytical instrumentation including ion chromatography or gas chromatography coupled to mass spectrometry, respectively [1,2]. These methods, however, require rather complex sample pretreatment and include a time-consuming separation step. Experimenteller Teil In this work, we eliminate sample preparation and perform direct analysis of model LIB samples by using ambient desorption/ionization high-resolution mass spectrometry (ADI-HR-MS). Based on previous work by our group [3], desorption/ionization of electrolytes is facilitated with a home-built low-temperature plasma probe (LTP, based on the original work by Harper et al. [4]) followed by mass spectrometric detection and identification. Commercially available electrolytes LP30 (composed of ethylene carbonate, dimethyl carbonate, and LiPF 6) and LP50 (composed of ethylene carbonate, ethyl methyl carbonate, and LiPF6) were investigated in thermal degradation experiments and results will be discussed. Ergebnisse After careful optimization of LTP-HR-MS and controlled aging of model electrolytes, new chemical species are identified that were not observed in our previous work. In addition, the influence of the electrolyte-additive vinylene carbonate on the LIB thermal aging process is investigated. Finally, a cooling system for the HR-MS sampling/ionization source stage will be presented. Advantages of temperature-controlled sampling will be discussed with a focus on very volatile compounds. Based on our results, LTP-HR-MS is considered a useful and complementary tool for future degradation studies on LIB electrolytes and electrodes. Neuer Aspekte Temperature-controlled degradation studies of electrolyte/additive mixtures for LIB with LTP-HR-MS; cooling/heating sampling plate for temperature-controlled sampling of volatile compounds. Referenzen [1] L. Terborg, S. Weber, F. Blaske, S. Passerini, M. Winter, U. Karst and S. Nowak, J. Power Sources 2013, 242, 832-837.; [2] L. Terborg, S. Weber, S. Passerini, M. Winter, U. Karst and S. Nowak, J. Power Sources 2014, 245, 836-840.; [3] B. Vortmann, S. Nowak and C. Engelhard, Anal. Chem. 2013, 85, 3433-3438.; [4] J. D. Harper, N. A. Charipar, C. C. Mulligan, X. Zhang, R. G. Cooks and Z. Ouyang, Anal. Chem. 2008, 80, 90979104.
Automated top-down mass spectrometry of hemoglobin for a clinical application Coelho Graça, Didia (1); Acosta-Martin, Adelina E. (1,2); Jabs, Wolfgang (3); Hartmer, Ralf (3); Clerici, Lorella (2); Meyer, Markus (3); Samii, Kaveh (4); Tsybin, Yury O. (5); Hochstrasser, Denis (1,2); Lescuyer, Pierre (1,2); Scherl, Alexander (1,2); Centnar, Zoltan (3) 1: Department of Human Protein Sciences, Faculty of Medicine, Geneva University, Geneva, Switzerland; 2: Department of Genetic and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland; 3: Bruker Daltonik GmbH, Bremen, Germany; 4: Division of Hematology, Geneva University Hospital, Geneva, Switzerland; 5: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Stichworte: Hemoglobin, ion trap, top-down, ETD Einleitung Hemoglobin (Hb) is a tetrameric protein present in red blood cells. Healthy adults have HbA (~97%), HbA2 (~3%) andHbF (98%.
Evaluation of native cross-links in elastin Schräder, Christoph U.; Heinz, Andrea; Schmelzer, Christian E.H. MLU Halle-Wittenberg, Deutschland Stichworte: native elastin, cross-linking, desmosine, lysinonorleucine, diagnostic ions Einleitung Elastin is a major protein of the extracellular matrix in gnathostomata and provides resilience and elasticity to numerous tissues including lung, skin and large blood vessels [1]. At the beginning of life, it is secreted as the soluble precursor protein tropoelastin followed by extensive cross-linking through reactive allysine residues yielding a highly hydrophobic biopolymer. So far the exact pattern of cross-linking remains unknown. Recent development allows the direct detection of peptides carrying the tetrafunctional cross-linking amino acids desmosine (DES) and isodesmosine (IDES) in an LC-MS based workflow [2]. This study aims to determine exact domains, which are connected to each other via bi- and tetrafunctional cross-linking amino acids. This provides indirect insight into the structural organization of mature elastin. Experimenteller Teil Isolation of bovine aortic elastin from one adult animal was carried out as described previously [3]. Purified elastin was digested by adding pancreatic elastase. Enzymatic digests were subsequently analyzed on a nanoHPLC - Orbitrap Fusion Tribrid mass spectrometer. For enriching cross-linked peptides, fractions were further collected applying a linear gradient on a reversed phase Agilent 1100 system. Relevant fractions were measured on an Orbitrap Velos Pro mass spectrometer. For detection of bi- and tetrafunctionally cross-linked peptides, processed data files were exported in MGF-format and analyzed with StavroX software [4] and PolyLinx software [5], respectively. Ergebnisse A total number of 310 linear elastin peptides were identified after digestion with PE, which corresponds to a sequence coverage of 78 %. It is interesting to note that many of the assumed cross-linking domains were found to be non-crosslinked as well. Search algorithms specifically designed for the detection of bi- and tetrafunctional amino acids allowed the detection of 41 bifunctionally cross-linked peptides. They were found to be inter- and intramolecularly crosslinked via lysinonorleucine (LNL) and allysine aldol (AA). It became apparent that some domains were cross-linked via LNL, whereas other domains were exclusively connected via AA. Since the digestion of elastin with site specific proteases, e.g. trypsin, is not possible, sequencing is more complicated. Thus the results were confirmed by manual spectrum interpretation. Interestingly, DES/IDES were found to connect two or three different peptide chains, although it was stated that they only may link two chains together. Moreover in an energy resolved study, it was found that the cross-linking amino acid LNL, one of the most abundant cross-linking amino acids in elastin, does not yield any diagnostic ions in fragment spectra but only fragment ions, which may also be produced upon fragmentation of lysine. This further impedes identification of those peptides. Mass spectrometric identification of peptides putatively cross-linked by LNL, therefore, requires high mass accuracy in terms of both MS and MS/MS mode. Neuer Aspekte Modern mass spectrometric methods allow insight into exact cross-linking sites in native elastin Referenzen [1] Mithieux, S. M., and Weiss, A. S. (2005) Elastin. Adv Protein Chem 70, 437-461; [2] Schräder, C. U., Heinz A., Majovsky, P., and Schmelzer, C. E. H. (2015) Fingerprinting desmosine-containing elastin peptides. J Am Soc Mass Spectrom doi: 10.1007/s13361-014-1075-9; [3] Schmelzer, C. E., Jung, M. C., Wohlrab, J., Neubert, R. H., and Heinz, A. (2012) Does human leukocyte elastase degrade intact skin elastin? FEBS J 279, 4191-4200;
[4] Gotze, M., Pettelkau, J., Schaks, S., Bosse, K., Ihling, C. H., Krauth, F., Fritzsche, R., Kuhn, U., and Sinz, A. (2012) StavroX--a software for analyzing crosslinked products in protein interaction studies. J Am Soc Mass Spectrom 23, 76-87; [5] Heinz, A., Ruttkies, C. K., Jahreis, G., Schrader, C. U., Wichapong, K., Sippl, W., Keeley, F. W., Neubert, R. H., and Schmelzer, C. E. (2013) In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials. Biochim
Influence of glycoforms on the tryptic digestion efficiency of immunoglobulin G based biopharmaceuticals Falck, David (1); Plomp, Rosina (1); Jansen, Bas J. (1); Reusch, Dietmar (2); Haberger, Markus (2); Wuhrer, Manfred (1,3) 1: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; 2: Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany; 3: Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands Stichworte: Bottom-up proteomics, Glycoform profiling, Biopharmaceuticals, nanoLC–MS, Tryptic digestion biases Einleitung Bottom-up proteomics is employed frequently in biological and clinical research. Nowadays, it is becoming increasingly popular for the analysis of biopharmaceuticals in discovery, development and batch control. The digestion of the target protein by proteases, generally by trypsin, into more easy to analyse peptides is a key step in bottom-up proteomics. Thus, a robust analysis needs to incorporate an efficient and unbiased tryptic digestion step. It has been observed previously that incomplete tryptic digestion can introduce biases into glycoform profiles[1]. Therefore, we investigated the preferential tryptic digestion of glycoforms of two biopharmaceutical formats: 1.an immunoglobulin G1(IgG1) monoclonal antibody(mAb) and 2.Flebogamma, an intravenous IgG(IVIG) from healthy donor pools. Via denaturation experiments, we collected evidence for the 3D-structure dependence of the biases. Experimenteller Teil The tryptic digestion of the target protein into glycopeptides was followed over time. The digestion of protein with an intact 3D-structure was compared to that of protein denatured by various methods. These included denaturation, reduction and alkylation as a gold standard as well as a novel, save and efficient protocol for IgG-denaturation. Glycopeptides from the conserved glycosylation site in the FC-part of IgG were detected by a nanoLC–ESI-q-TOF-MS method[2]. In addition, the glycoproteins and glycopeptides of partially digested samples were separated by gel electrophoresis and the residual glycoproteins were reanalysed after complete in-gel tryptic digestion. By relative quantitation of the glycopeptides and estimation of their absolute abundances, we could visualize a preference of trypsin for the digestion of certain glycoforms. Ergebnisse In general, we proved that the glycoforms of the IgG1 mAb containing high mannose and hybrid type glycans in the Fc portion were preferentially digested into the glycopeptides compared to those glycoforms containing complex type glycans. The biases observed in IVIG were smaller, because this formulation did not contain high mannose or hybrid glycans. The main bias in IVIG was created by preferential digestion of bisected species. Interestingly, the α2-3 linked sialic acid containing species in the IgG1 mAb were amongst the first to be digested while the α2-6 linked sialic acid containing species in IVIG were not preferred. Comparing the tryptic digestions of non-denatured and denatured protein revealed that the biases are much less pronounced in denatured samples. Additional experiments proved that this effect is independent of the increased digestion efficiency. A significant part of the digestion biases must therefore be caused by the differences in antibody 3D-structure induced by the different glycoforms. Our simple, novel pre-treatment method — acid incubation and evaporation — exhibited a denaturation efficiency comparable to the gold standard (see Methods) for IgG while significantly reducing the hands-on time and the toxicity of the reagents involved. It additionally offers the advantage of performing the tryptic digestion under optimal buffer conditions. In conclusion, proper denaturation and digestion completeness are key to avoiding biases in bottom-up proteomics based glycoform profiling. However, the digestion biases have implications beyond glycoproteomics. For example, relative protein quantitation (even with isotopically labelled standards) in two samples could be biased by differences in glycoform profiles, if complete tryptic digestion is not achieved. Neuer Aspekte
Trypsin preferentially digests certain glycoforms of IgG. This preference is extensively induced by the specific 3D structure of the glycoform. Referenzen [1] [1] Du et al. (2012), Journal of Chromatography B, 907:87-93; [2] [2] Selman et al. (2012), Journal of Proteomics 75(4):1318-1329
Seeking marker peptides for tracking age-related changes in elastic tissues Mora-Huertas, Angela Cristina; Schmelzer, Christian E.H.; Schräder, Christoph U.; Neubert, Reinhard H.H.; Heinz, Andrea Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale) Stichworte: elastin, cleavage susceptibility, aging, label free quantification Einleitung Elastic fibers are extracellular assemblies that provide elasticity and resilience to many tissues such as aorta and skin [1]. Reduced fiber strength and disorganization as a consequence of aging is an important cause of morbidity and mortality [2,3]. Elastin, as the core component of elastic fibers, is an insoluble and extremely durable protein that undergoes very little turnover. However, elastin isolated from skin samples of differently aged individuals shows striking differences in ultrastructure and cleavage susceptibility [4]. To understand the processes that lead to these structural changes, it is necessary to gain insight into cleavages that take place in vivo. In the present study, label-free quantification (LFQ) was applied to identify marker peptides that allow monitoring the aging of elastin. Experimenteller Teil Elastin was isolated from non-sun-exposed skin samples of differently aged individuals using a recently developed method [4]. Purified elastin samples were dried, weighed and digested using leukocyte (HLE) or pancreatic elastase (PE). The digests were analyzed under identical experimental conditions by LC-MS using an Ultimate nanoHPLC system (Thermo Fisher, Germany) coupled to a Q-TOF-2 mass spectrometer (Waters, UK). The raw files were analyzed using the LFQ module of the proteomics software PEAKS 7 (Bioinformatics Solutions Inc., Canada). Ergebnisse Elastin isolated from skin samples of all ages was readily digested by PE, as proven by LC-MS/MS analysis. Interestingly, HLE was only able to degrade elastin derived from elderly people even though it is considered to be an elastase. Digestion of intact fibers was not possible as indicated by the presence of a remaining yellowish elastin pellet in the reaction tube at the end of the incubation time. Moreover, no peptides were identified in the supernatant. Increasing the concentration of HLE and the incubation time of the digestion, respectively, did not change these findings. This suggests that the presence of impairments in the elastin fibers in matured tissues enhances the susceptibility of elastin to HLE. PE digests of elastin derived from differently aged people were further used to investigate whether differences may exist in the relative abundance of certain peptides. Preliminary results gained by LFQ analysis show age-related differences in the peptide patterns. Such differences include the complete absence of certain peptide species as well as distinct quantitative variationsand are most likely a result of cleavages introduced by extracellular proteases throughout life. Therefore, such peptides can serve as marker molecules to follow the age-related breakdown of elastin. Neuer Aspekte LFQ enables the identification of marker peptides that provide insights into the aging of elastin Referenzen [1] S.M. Mithieux, A.S. Weiss, Elastin, Adv. Protein Chem. 70 (2005) 437-461.; [2] Debelle L & Tamburro AM (1999) Elastin: molecular description and function. Int J Biochem Cell Biol 31, 261– 272.; [3] Sherratt M.J. Tissue elasticity and the ageing elastic fibre AGE 2009 31:305-325; [4] Schmelzer, C. E. H., Jung, M. C., Wohlrab, J., Neubert, R. H. H., and Heinz, A. (2012) Does human leukocyte elastase degrade intact skin elastin? FEBS J 279, 4191-4200
Protein structure prediction guided by crosslinking constraints – a systematic evaluation of the impact of the crosslinking spacer length Hofmann, Tommy (1); Fischer, Axel (2); Meiler, Jens (2); Kalkhof, Stefan (1) 1: Helmholtz-Zentrum für Umweltforschung, Deutschland; 2: Center for Structural Biology, Vanderbilt University, Nashville Stichworte: crosslinking, 3D-MS, mass spektrometry, computational biology Einleitung Driven by the mass spectrometry (MS) instrument development chemical crosslinking combined with MS becomes a powerful approach to obtain structural information about proteins and protein complexes [1]. Even though the number and resolution of distance restraints is typically insufficient to unambiguously determine a protein’s structure, it has been shown frequently that distance restraints obtained by chemical crosslinking can be used to refine protein models, restrict the protein fold space, guide protein/protein docking, and help distinguishing accurate from inaccurate structural models [2, 3]. However, the questions (i) what is the most useful crosslinker length to study a given protein, (ii) which model accuracy can be expected, and (iii) to which extend do crosslinks improve model selection are not yet fully evaluated. Experimenteller Teil In this study the number of crosslinks as well as the discriminative power of the derived distance restraints was systematically analyzed in silico on a set of 2055 non-redundant protein folds considering homobifunctional amine reactive crosslinkers between 1 and 60 Å [4]. Depending on the size of the proteins an optimal crosslinker length was predicted taking the total number of helpful crosslinks – i.e. crosslinks that significantly reduce search space – as well as the ratio of helpful vs possible crosslinks into account. Two protein structures had been de novo modeled with experimental restrains and our proposed optimal crosslinks and the enrichment as RMSD of both data sets compared. Ergebnisse We found that in average the data obtained by the optimal crosslinker improved the de novo modelling accuracy from 6.6 Å to 5.6 Å whereas utilization of the data of all five crosslinkers improved the modelling accuracy to 5.2 Å. Furthermore, restraints from crosslinking allowed for the selection of more native-like models. Whereas only 11% of the most accurate models could be selected with the default BCL::Fold scoring function, the value almost doubled to 21% when crosslinking data with the optimal crosslinker length were included. By including data obtained from all crosslinker lengths this value could be further improved to 24%. Finally, to study the effects of experimentally derived chemical cross-links on de-novo folding Cytochrome C and Oxymyoglobin were studied using published crosslinking resultsranging from 7.7 to 11.4 Å. Even though just a fraction of the theoretical and experimental crosslinks coincided model accuracy and discriminative power the scoring function improved significantly. In conclusion, for >70% of the low molecular weight proteins ( 0.95) was achieved by all methods for the BSA concentration series, with the best correlation given by OpenMS top three (R2 = 0.99). Also, relative standard deviations were < 0.1 on the log scale (< 25% on linear scale) for enolase and the three HeLa proteins for all methods. The EventDetector in Proteome Discoverer found a significantly higher number of features (2704) than the FeatureFinder in OpenMS (724). However, the percentage of low-variance features was higher with the OpenMS algorithm (78%; standard deviation < 0.1) compared to the Proteome Discoverer algorithm (48%). With the latter, a higher number of features will have to be disregarded due to their high variance in the replicates so the actual numbers will approximate. All methods tested were able to give a good estimate for the protein abundance in the model mixture. However, in clinical samples, the outcome of the methods was observed to vary considerably. Further studies should be performed to carefully assess and optimize the performance of the different label-free quantification methods available, so that reliability and reproducibility of these kinds of experiments will improve. Neuer Aspekte Comparison of label-free quantification approaches on a defined mixture of proteins. Referenzen
[1] Weisser H, Nahnsen S, Grossmann J, Nilse L, Quandt A, Brauer H, Sturm M, Kenar E, Kohlbacher O, Aebersold R, and Malmstrom L: An automated pipeline for high-throughput label-free quantitative proteomics. Journal of proteome research 12: 1628-1644, 2013.
Investigation of amino acid mutations in the plasma protein C1-Esterase-Inhibitor in a human population Steffen, Pascal Universitätsklinikum Hamburg-Eppendorf, Deutschland Stichworte: Glycopeptide, C1-inhibitor, Population Proteomics, Off-Gel Einleitung In the past years LC-MS/MS (especially triple-quadrupole mass spectrometer) is used to quantify protein disease markers. Here usually a unique peptide of the target protein is selected for monitoring (SRM)[1]. A further advancement in this field is the SISCAPA[2] process. Using immobilized anti-peptide antibodies target peptides are enriched resulting in a detection limit of proteins in the low ng/mL range. A problem that should be addressed is the variety of protein species in the population[3]. Some of these species include different PTMs and/or point mutations, which result in different m/z values. Tryptic peptides containing these changes are lost for SRM. This study investigates if these variances are critical for the quantification of defined proteins in human blood plasma by SRM. Experimenteller Teil The therapeutic protein “Berinert®” from CSL Behring, containing C1-Esterase-Inhibitor, was dissolved to obtain a stock solution of 6.5 mg/mL. Enrichment of Glycopeptides using HILIC: Tryptic peptides of C1-Esterase-Inhibitor were fractionated on a self made mini column packed with TSKGel® Amide 80. Peptide fractionation: Tryptic peptides of C1-Esterase-Inhibitor were incubated with PNGase F for removing Nglycans and then subjected to a gel-free isoelectric focussing (OFF-gel). Mass spectrometric analysis of the fractions: All fractions were analysed on a LC-MS/MS instrument (Thermo FusionTM using an HCDpdETD setting with prior online separation on an Acclaim® PepMap 100 C18 Nano-Trap column). Data processing and interpretation was performed with severeal algorithms (ProteomeDiscoverer, Sequest TM, X!Tandem and ByonicTM).
Ergebnisse After measuring the flowthrough and the eluate of the HILIC enrichment, using HCDpdETD setting, it could be shown that the glycopeptides were quantitatively separated from the non-glycopeptides. Using Byonic with 2 allowed missed cleavages, 10 ppm precursor mass tolerance and 0.1 Da and 0.6 Da fragment mass tolerance for HCD and ETD respectively, carbamidomethyl (C) as fixed and oxidation (M) as common modifications and a list of common human plasma N-glycans and 98 common O-glycans a total of 4 N-glycans and 1 O-glycan (with some degree of microheterogeneity) were identified. It was hypothesised that using isoelectric focussing peptides including an amino acid exchange because of a mutation will be detectable, resulting in signals for which no sequence assignment is possible by search engines. After analyzing the 24 fractions of the isoelectric focusing with several search engines nearly all signals above a threshold of 5% were assigned to peptides of the C1-Inhibitor yielding a sequence coverage of 83.8% (semi-tryptic search). For identification of peptides of the C1-inhibitor containing mutations, ProteomeDiscovererTM was used to export all unidentified MS/MS-spectra. These spectra were then analyzed using ByonicTM with a list of all possible amino acid mutations. By this approach 349 mutations were identified. Considering that the C1-Inhibitor was purified from a pool of more than 6000 donors and that the intensities of the peptides with amino acid mutations were less than 1% of those corresponding to the wild-type it seems fair to say that these mutations will not have much influence on the quantitation of protein marker but at the same time, to circumvent this statistical problem, two or more unique peptides should be used to quantify a target protein by SRM. Neuer Aspekte
Screening for mutation present in tryptic peptides of C1-Esterase-Inhibitor yielded from a pool of more than 6000 human individuals. Referenzen [1] Gerber SA, Rush J, Stemman O, Kirschner MW, et al., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A, 2003. 100(12): p. 6940-5.; [2] Anderson NL, Jackson A, Smith D, Hardie D, et al., SISCAPA peptide enrichment on magnetic beads using an inline bead trap device. Mol Cell Proteomics, 2009. 8(5): p. 995-1005; [3] Nedelkov D, Kiernan UA, Niederkofler EE, Tubbs KA, et al., Investigating diversity in human plasma proteins. Proc Natl Acad Sci U S A, 2005. 102(31): p. 10852-7
Discovery of Pesticide Protomers Using Routine Ion Mobility Screening McCullagh, Michael (1); Turkovic, Davor (2); Neeson, Kieran (1); Goshawk, Jeff (1); Eatough, David (1); Carver, Chris (1) 1: Waters Corporation, UK; 2: Waters GmbH, Deutschland Stichworte: ion mobility, protomers, pesticides Einleitung Criteria to instill confidence in identification include acceptable product ion ratio tolerances and relative intensities of the detected ions, expressed as a percentage of the intensity of the most intense (abundant) ion or product ion, which should correspond to those of the calibration standard at comparable concentrations and measured under the same conditions. Ion ratio performance can vary with instrumentation, matrix and is affected by sample concentration. SANCO/12571/2013 guidance document describes the method validation and analytical quality control requirements to support the validity of data used for checking compliance with maximum residue limits, enforcement actions, or assessment of consumer exposure to pesticides in the EU. Here we use ion mobility mass spectrometry to gain greater understanding of ion ratio variation. Experimenteller Teil The assay is based on the analysis of sample extracts and matrix matched calibrants of pear, ginger, leek and mandarin, as well as quality control samples generated for an EU-RL proficiency test.These samples were analysed using ultra performance liquid chromatography using a UPLC® BEH C18 (1.7µm, 2.1x100 mm) analytical column. In addition a series of mixtures of pesticide solvent standards were injected on column. The chromatographic conditions were comprised of a 17 minute water/acetonitrile (0.1% Formic Acid) gradient at 0.45 ml/min and sample injection volumes of 5µl were used. Positive ion electrospray with ion mobility mass spectrometry data acquisition was performed using a Synapt G2-S mass spectrometer. Ergebnisse Empirically isobaric pesticide protomers have been identified and characterised using ion mobility. It has been possible to separate the protomers (ions different only by their protonation site), determine their respective collision cross section and individual protomer fragmentation dissociation pathways. This has enabled unique visibility of product ion formation information, enabling the product ions to be selected that will result in improved product ion ratio reproducibility. For the assay, UPLC-HDMSE experiments were performed on a Synapt G2-S using a series of standard solutions, spiked matrices and a previous proficiency test.
For indoxacarb determined to be present in proficiency sample FV-13, two mobility separated species, with CCS values of 136.49 Ų and 147.94 Ų were obtained. From the structure of indoxacarb it can be seen that protonation is possible on more than one site and that multiple sites of protonation may have been observed, and two protomers have been mobility resolved. The fragmentation spectra generated from the mobility separated protomers of indoxacarb allowed the distinctive different fragments of each respective protomer to be identified, as well as common fragments (m/z 190 and m/z 249). A further example of protomer formation observed, is for fenpyroximate with measured CCS values of 147.15 Ų and 158.33 Ų. The protomers of fenpyroximate each respectively produce one of the two most abundant fragment ions. If conventional MRM analysis was performed the ion ratio measure would be affected by protomer formation. It has previously been observed that protomer formation is affected by experimental parameters such as flow rate, matrix, cone voltage and capillary voltage. As a result the fragments observed will be affected by these parameters and will affect the reproducibility of the fragments and their intensities observed. This in turn could have impact upon assays where by MRM transition is the method of choice for confirmation analysis Neuer Aspekte Identification and collision cross section measurement of empirically isobaric pesticide protomers in residue analysis.
Using the Routine Separation Dimension and Identification Criteria of UPLC Ion Mobility to Enhance Specificity in Profiling Complex Samples. McCullagh, Michael (1); Turkovic, Davor (2); Thomas, Christoph (2); Neeson, Kieran (1); Goshawk, Jeff (1); Carver, Christopher (1); Douce, David (1) 1: Waters Corp., UK; 2: Waters GmbH, Deutschland Stichworte: CCS, ion mobility, passiflora, glycosides Einleitung The combined peak capacity of UPLC/ion mobility and collision cross section mesurements (CCS) can be used to produce routine unequivocal identification of marker flavonoid isomers in complex mixtures such as herbal tea products (functional foods)/ phytomedicines). The genus Passiflora comprises of approximately 450 species, but only a few are commercially exploited. Several Passiflora (Passifloraceae) species are utilized as phytomedicines (sedative/tranquillising), the species contain flavonoids, mainly C-glycosylflavones (apigenin and luteolin derivatives; frequently occurring as isomers). Flavonoids are one of the largest and most wide spread classes of compounds and possess diverse pharmacological and biological properties. “Natural” is not neccessarily safe, but such products can be if produced to legisative standards e.g. (Directive 2004/24/EC, which came into full effect 30 April 2011). Experimenteller Teil Hydroethanolic extracts of P.incarnata, P.edulis, P.caerulea and P.alata were analysed using ultra performance liquid chromatography. A UPLC® BEH C18 (1.7µm, 2.1x100 mm) analytical column was utilised. The chromatographic conditions were comprised of a 17 minute water + (0.1% Formic Acid) /acetonitrile + (0.1% Formic Acid) gradient at 0.75 ml/min with a sample injection volume of 2µl. Negative ion electrospray with ion mobility HDMSE data acquisition was performed using a Synapt G2-S mass spectrometer. Ergebnisse Collision cross sections (CCS), accurate mass, fragment ions and retention time have been used to profile the hydroethanolic extracts of P. incarnata, P. alata, P. edulis and P. caerulea, grown in Brazil. This approach offers a unique selectivity in profiling complex mixtures. Results obtained clearly show the benefits of using the collisions cross section measurements and the combined peak capacity of UPLC with ion mobility. The enhanced peak capacity enabled more information to be extracted from fragmentation studies and the individual fragmentation spectra have been obtained for flavonoid isomers which are co-eluting. From the extracts characteristic assignment for 6-C and 8-C flavonoid glycosides isomers (vitexin and isovitexin) (orientin and isoorientin) were obtained.
Collision cross section measurements were obtained for the marker flavonoid standards, and this information was used to create a scientific library incorporating the expected CCS values. The four Passiflora extracts were analysed and routinely screened against the flavonoid CCS library, to determine the presence/unequivocal identification of the 6-C and 8-C flavonoid glycosides isomers. CCS measurements for marker glycoside pairs (vitexin and isovitexin) 188.8 Ǻ2/195.5Ǻ2 have been determined. For (orientin and isoorientin) 187.7 Ǻ2/198.1 Ǻ2 were obtained. This proved that it is possible to distinguish the marker isomer pairs for the extracts analysed using CCS measurements. When comparing the expected and the measured collision cross sections, the CCS measurement errors were typically