M Ethods in M Olecular B Iology

M ETHODS IN M OLECULAR B IOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the ﬁrst to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed. Quantitative Methods in Proteomics

Second Edition

Edited by Katrin Marcus and Martin Eisenacher

Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-Universit€at Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany Barbara Sitek

Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-Universit€at Bochum, Bochum, Germany; Klinik für An€asthesiologie, Intensivmedizin und Schmerztherapie, Universit€atsklinikum Knappschaftskrankenhaus Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany Editors Katrin Marcus Martin Eisenacher Medizinisches Proteom-Center (MPC) Medizinisches Proteom-Center (MPC) Medical Faculty Medical Faculty Ruhr-Universit€at Bochum Ruhr-Universit€at Bochum Bochum, Germany Bochum, Germany Medical Proteome Analysis Medical Proteome Analysis Center for Proteindiagnostics (PRODI) Center for Proteindiagnostics (PRODI) Ruhr-University Bochum Ruhr-University Bochum Bochum, Germany Bochum, Germany

Barbara Sitek Medizinisches Proteom-Center (MPC) Medical Faculty Ruhr-Universit€at Bochum Bochum, Germany Klinik fu¨r An€asthesiologie Intensivmedizin und Schmerztherapie Universit€atsklinikum Knappschaftskrankenhaus Bochum Bochum, Germany Medical Proteome Analysis Center for Proteindiagnostics (PRODI) Ruhr-University Bochum Bochum, Germany

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-1023-7 ISBN 978-1-0716-1024-4 (eBook) https://doi.org/10.1007/978-1-0716-1024-4

© Springer Science+Business Media, LLC, part of Springer Nature 2021 Chapters 1, 8, 16, 18, 25 and 26 are licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapters. This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Preface

The main approach and structure of the book have been retained in the second edition, but some methodical updates and new aspects in data analysis have been added. Today, protein identification is an almost routine requirement. However, reliable techniques for quantifying unmodified proteins (including those that escape detection under standard conditions, such as protein isoforms and membrane proteins) as well as the detection and quantification of post-translational protein modifications is still far from being a matter of routine. Hence, there is a need for a profound understanding of the principles underlying modern protein analysis, in order to apply and improve established and novel methods successfully. In the eight years, since the first edition of this book was published the data analysis and digitalization became increasingly important. Even in the public and media discussions, it is nowadays commonly accepted that digitalization is part of each and every area of life. This is of course true for science and has been lived reality in the mass spectrometry community since its beginning. It is obligatory that spectra are identified and quantified with algorith- mic methods from computer science and that peptides/protein abundances are assessed for significance with methods from statistics. This new edition gives a detailed survey of Quantitative Methods in Proteomics, addressing topics and methods from statistical issues when planning proteomics experiments (Chap. 1), new chapters dealing with protein quantification as a basis for realization of quantitative studies (Chaps. 2 and 3), gel-based (Chaps. 4–7), and mass spectrometry-based quantification techniques as TMT, IPTL, PRM, and MALDI Imaging (Chaps. 8–12). In this edition, the main meaning has been given to application of different methods as possibility for the readers to get a wide overview on how proteomics quantification can be used in diverse questions. In these chapters (Chaps. 13–26), different techniques (e.g., TMT, SILAC, PTM analysis, DIA, cross-linking) are applied for various kinds of samples (e.g., human, yeast, bacteria, cell culture samples). The subsequent section is addressing the up-to-date topics of software and data analysis (Chaps. 27–31). As the peptide undersampling (low coverage) is one of the most important challenges of Proteomics, a specific chapter about missing value monitoring is dealing with this. Two chapters are dedicated to give a meaning to pure numbers: one about variance- sensitive clustering that helps to find probably unknown structure in the data and the other about network meta-analysis that helps to compare own data to previously published data although having another experimental group structure. As the guest editors of this volume Quantitative Methods in Proteomics, we would like to thank all of the authors and coauthors for sharing their experience, knowledge, and time to make this new edition possible. We hope that the reader will take advantage of his/her research work from this comprehensive and competent overview of the important and still growing field of quantitative proteomics. Enjoy!

Bochum, Germany Katrin Marcus Martin Eisenacher Barbara Sitek

v Contents

Preface ...... v Contributors...... xi

1 Important Issues in Planning a Proteomics Experiment: Statistical Considerations of Quantitative Proteomic Data...... 1 Karin Schork, Katharina Podwojski, Michael Turewicz, Christian Stephan, and Martin Eisenacher 2 Good Old-Fashioned Protein Concentration Determination by Amino Acid Analysis ...... 21 Caroline May, Bettina Serschnitzki, and Katrin Marcus 3 Protein Quantification Using the “Rapid Western Blot” Approach ...... 29 Katalin Barkovits, Kathy Pfeiffer, Britta Eggers, and Katrin Marcus 4 The Whereabouts of 2D Gels in Quantitative Proteomics ...... 41 Thierry Rabilloud and Cećile Lelong 5 Proteome Analysis with Classical 2D-PAGE ...... 53 Caroline May, Frederic Brosseron, Kathy Pfeiffer, Kristin Fuchs, Helmut E. Meyer, Barbara Sitek, and Katrin Marcus 6 Silver Staining of 2D Electrophoresis Gels ...... 63 Thierry Rabilloud 7 Differential Proteome Analysis Using 2D-DIGE ...... 77 Caroline May, Frederic Brosseron, Piotr Chartowski, Kristin Fuchs, Helmut E. Meyer, Barbara Sitek, and Katrin Marcus 8 Quantitative Mass Spectrometry-Based Proteomics: An Overview...... 85 Svitlana Rozanova, Katalin Barkovits, Miroslav Nikolov, Carla Schmidt, Henning Urlaub, and Katrin Marcus 9 Tandem Mass Tags for Comparative and Discovery Proteomics...... 117 Oliver Pagel, Laxmikanth Kollipara, and Albert Sickmann 10 An Approach for Triplex-IPTL ...... 133 Christian J. Koehler and Bernd Thiede 11 Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM) ...... 145 Katalin Barkovits, Weiqiang Chen, Michael Kohl, and Thilo Bracht 12 Quantitative Approach Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) Mass Spectrometry...... 159 Brooke A. Dilmetz, Peter Hoffmann, and Mark R. Condina 13 Application of SILAC Labeling in Phosphoproteomics Analysis...... 167 Markus Stepath and Thilo Bracht

vii viii Contents

14 Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy ...... 185 Ivan Silbern, Pan Fang, Yanlong Ji, Lenz Christof, Henning Urlaub, and Kuan-Ting Pan 15 High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry ...... 205 Kaiwen Yu, Zhen Wang, Zhiping Wu, Haiyan Tan, Ashutosh Mishra, and Junmin Peng 16 Quantification and Identification of Post-Translational Modifications Using Modern Proteomics Approaches ...... 225 Anja Holtz, Nathan Basisty, and Birgit Schilling 17 Affinity Enrichment Chemoproteomics for Target Deconvolution and Selectivity Profiling...... 237 Thilo Werner, Michael Steidel, H. Christian Eberl, and Marcus Bantscheff 18 2nSILAC for Quantitative Proteomics of Prototrophic Baker’s Yeast ...... 253 Stefan Dannenmaier, Silke Oeljeklaus, and Bettina Warscheid 19 Metabolic Labeling of Clostridioides difficile Proteins...... 271 Anke Trautwein-Schult, Ju¨rgen Bartel, Sandra Maaß, and Do¨rte Becher 20 Application of Label-Free Proteomics for Quantitative Analysis of Urothelial Carcinoma and Cystitis Tissue...... 283 Kathrin E. Witzke, Frederik Großerueschkamp, Klaus Gerwert, and Barbara Sitek 21 Quantitative MS Workflow for a High-Quality Secretome Analysis by a Quantitative Secretome-Proteome Comparison ...... 293 Gereon Poschmann, Nina Prescher, and Kai Stu¨hler 22 Establishing a Custom-Fit Data-Independent Acquisition Method for Label-Free Proteomics ...... 307 Britta Eggers, Martin Eisenacher, Katrin Marcus, and Julian Uszkoreit 23 Label-Free Proteomics of Quantity-Limited Samples Using Ion Mobility-Assisted Data-Independent Acquisition Mass Spectrometry...... 327 Ute Distler, Malte Sielaff, and Stefan Tenzer 24 DIA-MSE to Study Microglial Function in Schizophrenia ...... 341 Guilherme Reis-de-Oliveira, Victor Corasolla Carregari, and Daniel Martins-de-Souza 25 Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics...... 353 Andrew J. Percy and Christoph H. Borchers 26 Quantitative Cross-Linking of Proteins and Protein Complexes ...... 385 Marie Barth and Carla Schmidt 27 Missing Value Monitoring to Address Missing Values in Quantitative Proteomics...... 401 Vittoria Matafora and Angela Bachi Contents ix

28 Quantitative Proteome Data Analysis of Tandem Mass Tags Labeled Samples ...... 409 Oliver Pagel, Laxmikanth Kollipara, and Albert Sickmann 29 Mining Protein Expression Databases Using Network Meta-Analysis...... 419 Christine Winter and Klaus Jung 30 A Tutorial for Variance-Sensitive Clustering and the Quantitative Analysis of Protein Complexes...... 433 Veit Schwammle€ and Christina E. Hagensen 31 Automated Workﬂow for Peptide-Level Quantitation from DIA/SWATH-MS Data ...... 453 Shubham Gupta and Hannes Ro¨st

Index ...... 469 Contributors

ANGELA BACHI • IFOM, FIRC Institute of Molecular Oncology, Milan, Italy MARCUS BANTSCHEFF • Cellzome GmbH, GlaxoSmithKline, Heidelberg, Germany KATALIN BARKOVITS • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany JU¨ RGEN BARTEL • Department of Microbial Proteomics, Center for Functional Genomics of Microbes, Institute of Microbiology, University of Greifswald, Greifswald, Germany MARIE BARTH • Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle- Wittenberg, Halle, Germany NATHAN BASISTY • Buck Institute for Research on Aging, Novato, CA, USA DO¨ RTE BECHER • Department of Microbial Proteomics, Center for Functional Genomics of Microbes, Institute of Microbiology, University of Greifswald, Greifswald, Germany CHRISTOPH H. BORCHERS • Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, QC, Canada; Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, Russia THILO BRACHT • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-Universitat€ Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany; Klinik fu¨r Anasthesiologie,€ Intensivmedizin und Schmerztherapie, Universitatsklinikum€ Knappschaftskrankenhaus Bochum, Bochum, Germany FREDERIC BROSSERON • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany VICTOR CORASOLLA CARREGARI • Lab of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil PIOTR CHARTOWSKI • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany WEIQIANG CHEN • Medizinisches Proteom-Center (MPC), Ruhr-Universitat€ Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany LENZ CHRISTOF • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; Bioanalytics Group, Institute of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany MARK R. CONDINA • Future Industries Institute, University of South Australia, Adelaide, Australia STEFAN DANNENMAIER • Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany BROOKE A. DILMETZ • Future Industries Institute, University of South Australia, Adelaide, Australia

xi xii Contributors

UTE DISTLER • Institute of Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany; Research Center for Immunotherapy (FZI), University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany H. CHRISTIAN EBERL • Cellzome GmbH, GlaxoSmithKline, Heidelberg, Germany BRITTA EGGERS • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany MARTIN EISENACHER • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany PAN FANG • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany KRISTIN FUCHS • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-Universitat€ Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany; Klinik fu¨r Anasthesiologie,€ Intensivmedizin und Schmerztherapie, Universitatsklinikum€ Knappschaftskrankenhaus Bochum, Bochum, Germany KLAUS GERWERT • Biospectroscopy, Center for Proteindiagnostics (PRODI), Ruhr-Universitat€ Bochum, Bochum, Germany FREDERIK GROßERUESCHKAMP • Biospectroscopy, Center for Proteindiagnostics (PRODI), Ruhr-Universitat€ Bochum, Bochum, Germany SHUBHAM GUPTA • Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada CHRISTINA E. HAGENSEN • Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark PETER HOFFMANN • Future Industries Institute, University of South Australia, Adelaide, Australia ANJA HOLTZ • Buck Institute for Research on Aging, Novato, CA, USA YANLONG JI • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; Hematology/Oncology, Department of Medicine II, Johann Wolfgang Goethe University, Frankfurt, Germany KLAUS JUNG • Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany CHRISTIAN J. KOEHLER • Department of Biosciences, University of Oslo, Oslo, Norway MICHAEL KOHL • Medizinisches Proteom-Center (MPC), Ruhr-Universitat€ Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany LAXMIKANTH KOLLIPARA • Leibniz-Institut fu¨r Analytische Wissenschaften—ISAS—e.V., Bunsen-Kirchhoff-Straße, Dortmund, Germany CE´ CILE LELONG • Chemistry and Biology of Metals, Univ. Grenoble-Alpes, CNRS UMR5249, IRIG-DIESE-CBM, CEA Grenoble, Grenoble Cedex 9, France SANDRA MAAß • Department of Microbial Proteomics, Center for Functional Genomics of Microbes, Institute of Microbiology, University of Greifswald, Greifswald, Germany KATRIN MARCUS • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany Contributors xiii

DANIEL MARTINS-DE-SOUZA • Lab of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil; Instituto Nacional de Biomarcadores em Neuropsiquiatria (INBION), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnologico, Saõ Paulo, Brazil; Experimental Medicine Research Cluster (EMRC), University of Campinas, Campinas, Brazil; D’Or Institute for Research and Education (IDOR), Saõ Paulo, Brazil VITTORIA MATAFORA • IFOM, FIRC Institute of Molecular Oncology, Milan, Italy CAROLINE MAY • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany HELMUT E. MEYER • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany ASHUTOSH MISHRA • Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, TN, USA MIROSLAV NIKOLOV • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany SILKE OELJEKLAUS • Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany OLIVER PAGEL • Leibniz-Institut fu¨r Analytische Wissenschaften—ISAS—e.V., Bunsen-Kirchhoff-Straße, Dortmund, Germany KUAN-TING PAN • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; Hematology/Oncology, Department of Medicine II, Johann Wolfgang Goethe University, Frankfurt, Germany; Frankfurt Cancer Institute, Goethe University, Frankfurt am Main, Germany JUNMIN PENG • Departments of Structural Biology and Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA; Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, TN, USA ANDREW J. PERCY • Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA KATHY PFEIFFER • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany KATHARINA PODWOJSKI • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Bayer Schering Pharma AG, Wuppertal, Germany GEREON POSCHMANN • Institute of Molecular Medicine I, Proteome Research, University Hospital Du¨sseldorf, Heinrich Heine University Du¨sseldorf, Du¨sseldorf, Germany NINA PRESCHER • Institute of Molecular Medicine I, Proteome Research, University Hospital Du¨sseldorf, Heinrich Heine University Du¨sseldorf, Du¨sseldorf, Germany THIERRY RABILLOUD • Chemistry and Biology of Metals, Univ. Grenoble-Alpes, CNRS UMR5249, IRIG-DIESE-CBM, CEA Grenoble, Grenoble Cedex 9, France GUILHERME REIS-DE-OLIVEIRA • Lab of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil HANNES RO¨ ST • Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada SVITLANA ROZANOVA • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany xiv Contributors

BIRGIT SCHILLING • Buck Institute for Research on Aging, Novato, CA, USA CARLA SCHMIDT • Interdisciplinary Research Center HALOmem, Charles Tanford Protein Center, Institute for Biochemistry and Biotechnology, Martin Luther University Halle- Wittenberg, Halle, Germany KARIN SCHORK • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany € VEIT SCHWAMMLE • Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark BETTINA SERSCHNITZKI • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany ALBERT SICKMANN • Leibniz-Institut fu¨r Analytische Wissenschaften—ISAS—e.V., Bunsen-Kirchhoff-Straße, Dortmund, Germany; Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, UK; Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany MALTE SIELAFF • Institute of Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany; Research Center for Immunotherapy (FZI), University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany IVAN SILBERN • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; Bioanalytics Group, Institute of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany BARBARA SITEK • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany; Klinik fu¨r Anasthesiologie,€ Intensivmedizin und Schmerztherapie, Universitatsklinikum€ Knappschaftskrankenhaus Bochum, Bochum, Germany MICHAEL STEIDEL • Cellzome GmbH, GlaxoSmithKline, Heidelberg, Germany MARKUS STEPATH • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- Universitat€ Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany CHRISTIAN STEPHAN • KAIROS GmbH, Bochum, Germany; Medical Faculty, Ruhr- Universitat€ Bochum, Bochum, Germany KAI STU¨ HLER • Institute of Molecular Medicine I, Proteome Research, University Hospital Du¨sseldorf, Heinrich Heine University Du¨sseldorf, Du¨sseldorf, Germany HAIYAN TAN • Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital, Memphis, TN, USA STEFAN TENZER • Institute of Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany; Research Center for Immunotherapy (FZI), University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany BERND THIEDE • Department of Biosciences, University of Oslo, Oslo, Norway ANKE TRAUTWEIN-SCHULT • Department of Microbial Proteomics, Center for Functional Genomics of Microbes, Institute of Microbiology, University of Greifswald, Greifswald, Germany Contributors xv

MICHAEL TUREWICZ • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany HENNING URLAUB • Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany; Bioanalytics Group, Institute of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany; Hematology/ Oncology, Department of Medicine II, Johann Wolfgang Goethe University, Frankfurt, Germany JULIAN USZKOREIT • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany ZHEN WANG • Departments of Structural Biology and Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA BETTINA WARSCHEID • Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany THILO WERNER • Cellzome GmbH, GlaxoSmithKline, Heidelberg, Germany CHRISTINE WINTER • Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany KATHRIN E. WITZKE • Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr- University Bochum, Bochum, Germany; Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany ZHIPING WU • Departments of Structural Biology and Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA KAIWEN YU • Departments of Structural Biology and Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA