Analysis of Protein Post-Translational Modifications by Mass Spectrometry

John Griffiths is an analytical chemist with 30 years experience in the analysis of a wide range of analytes using mass spectrometry and other techniques. For the past 13 years, John has focused solely on the application of mass spectrometry to the analysis of peptides and proteins – proteomics. John has published multiple papers on biological mass spectrometry and has presented his work at both national and international conferences. John has a particular interest in the analysis of PTMs and has developed a number of strategies, such as the MIDAS with Richard Unwin, to enhance their detection. John is also the director of a mass spectrometry training and consultancy enterprise, MS-Insight Ltd. Richard Unwin is a biochemist and mass spectrometrist with over 18 years’ experience in the field of proteomics, in particular the quantification and characterization of proteins by mass spectrometry. Richard was among the first to develop the use of iTRAQ technology for protein quantitation and, with John Griffiths, was also amongst the first researchers to begin to realize the potential of multiple reaction monitoring MS for the discovery and characterization of post-translational modifications. Richard has contributed chapters on proteomics methods for a number of textbooks, aimed at both practicing mass spectrometrists and undergraduates, and has authored over 40 papers in the field.

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• List of Contributors xiPreface xv1 Introduction 1Rebecca Pferdehirt, Florian Gnad and Jennie R. Lill1.1 Post-translational Modification of Proteins 11.2 Global versus Targeted Analysis Strategies 31.3 Mass Spectrometric Analysis Methods for the Detection of PTMs 51.3.1 Data-Dependent and Data-Independent Analyses 61.3.2 Targeted Analyses 71.3.3 Multiple Reaction Monitoring 81.3.4 Multiple Reaction Monitoring Initiated Detection and Sequencing 91.4 The Importance of Bioinformatics 9Acknowledgements 11References 112 Identification and Analysis of Protein Phosphorylation by Mass Spectrometry 17Dean E. McNulty, Timothy W. Sikorski and Roland S. Annan2.1 Introduction to Protein Phosphorylation 172.2 Analysis of Protein Phosphorylation by Mass Spectrometry 252.3 Global Analysis of Protein Phosphorylation by Mass Spectrometry 392.4 Sample Preparation and Enrichment Strategies for Phosphoprotein Analysis by Mass Spectrometry 462.5 Multidimensional Separations for Deep Coverage of the Phosphoproteome 542.6 Computational and Bioinformatics Tools for Phosphoproteomics 572.7 Concluding Remarks 65References 663 Analysis of Protein Glycosylation by Mass Spectrometry 89David J. Harvey3.1 Introduction 893.2 General Structures of Carbohydrates 893.2.1 Protein-Linked Glycans 903.3 Isolation and Purification of Glycoproteins 943.3.1 Lectin Affinity Chromatography 953.3.2 Boronate-Based Compounds 953.3.3 Hydrazide Enrichment 963.3.4 Titanium Dioxide Enrichment of Sialylated Glycoproteins 963.4 Mass Spectrometry of Intact Glycoproteins 963.5 Site Analysis 963.6 Glycan Release 983.6.1 Use of Hydrazine 993.6.2 Use of Reductive β-Elimination 993.6.3 Use of Enzymes 1003.7 Analysis of Released Glycans 1023.7.1 Cleanup of Glycan Samples 1023.7.2 Derivatization 1023.7.2.1 Derivatization at the Reducing Terminus 1023.7.2.2 Derivatization of Hydroxyl Groups: Permethylation 1043.7.2.3 Derivatization of Sialic Acids 1063.7.3 Exoglycosidase Digestions 1063.7.4 HPLC and ESI 1073.8 Mass Spectrometry of Glycans 1073.8.1 Aspects of Ionization for Mass Spectrometry Specific to the Analysis of Glycans 1073.8.1.1 Electron Impact (EI) 1073.8.1.2 Fast Atom Bombardment (FAB) 1083.8.1.3 Matrix-Assisted Laser Desorption/Ionization (MALDI) 1083.8.1.4 Electrospray Ionization (ESI) 1133.8.2 Glycan Composition by Mass Spectrometry 1143.8.3 Fragmentation 1143.8.3.1 Nomenclature of Fragment Ions 1163.8.3.2 In-Source Decay (ISD) Ions 1163.8.3.3 Postsource Decay (PSD) Ions 1173.8.3.4 Collision-Induced Dissociation (CID) 1173.8.3.5 Electron Transfer Dissociation (ETD) 1183.8.3.6 Infrared Multiphoton Dissociation (IRMPD) 1183.8.3.7 MSn 1183.8.3.8 Fragmentation Modes of Different Ion Types 1193.8.4 Ion Mobility 1263.8.5 Quantitative Measurements 1283.9 Computer Interpretation of MS Data 1283.10 Total Glycomics Methods 1303.11 Conclusions 131Abbreviations 131References 1334 Protein Acetylation and Methylation 161Caroline Evans4.1 Overview of Protein Acetylation and Methylation 1614.1.1 Protein Acetylation 1614.1.2 Protein Methylation 1624.1.3 Functional Aspects 1634.1.4 Mass Spectrometry Analysis 1634.2 Mass Spectrometry Behavior of Modified Peptides 1644.2.1 MS Fragmentation Modes 1644.2.2 Acetylation- and Methylation-Specific Diagnostic Ions in MS Analysis 1654.2.3 Application of MS Methodologies for the Analysis of PTM Status 1684.2.4 Quantification Strategies 1694.2.4.1 Single Reaction Monitoring/Multiple Reaction Monitoring 1704.2.4.2 Parallel Reaction Monitoring 1714.2.4.3 Data-Independent Acquisition MS 1724.2.4.4 Ion Mobility MS 1734.2.5 Use of Stable Isotope–Labeled Precursors 1744.2.5.1 Dynamics of Acetylation and Methylation 1744.2.5.2 Stoichiometry of Acetylation and Methylation 1754.3 Global Analysis 1764.3.1 Top-Down Proteomics 1764.3.2 Middle Down 1774.4 Enrichment 1784.4.1 Immunoaffinity Enrichment 1784.4.2 Reader Domain-Based Capture 1794.4.2.1 Kac-Specific Capture Reagents 1794.4.2.2 Methyl-Specific Capture Reagents 1804.4.3 Biotin Switch-Based Capture 1804.4.4 Enrichment of N-Terminally Acetylated Peptides 1814.5 Bioinformatics 1814.5.1 Assigning Acetylation and Methylation Status 1824.5.2 PTM Repositories and Data Mining Tools 1834.5.3 Computational Prediction Tools for Acetylation and Methylation Sites 1834.5.4 Information for Design of Follow-Up Experiments 1854.6 Summary 185References 1855 Tyrosine Nitration 197Xianquan Zhan, Ying Long and Dominic M. Desiderio5.1 Overview of Tyrosine Nitration 1975.2 MS Behavior of Nitrated Peptides 1995.3 Global Analysis of Tyrosine Nitration 2085.4 Enrichment Strategies 2145.5 Concluding Remarks 221Acknowledgements 222Abbreviations 222References 2236 Mass Spectrometry Methods for the Analysis of Isopeptides Generated from Mammalian Protein Ubiquitination and SUMOylation 235Navin Chicooree and Duncan L. Smith6.1 Overview of Ub and SUMO 2356.1.1 Biological Overview of Ubiquitin-Like Proteins 2356.1.2 Biological Overview of Ub and SUMO 2366.1.3 Biological Functions of Ub and SUMO 2366.2 Mass Spectrometry Behavior of Isopeptides 2376.2.1 Terminology of a Ub/Ubl isopeptide 2376.2.2 Mass Spectrometry Analysis of SUMO-Isopeptides Derived from Proteolytic Digestion 2386.2.3 Analysis of SUMO-Isopeptides with Typical Full-Length Tryptic Iso-chains 2386.2.4 Analysis of SUMO-Isopeptides with Atypical Tryptic Iso-chains and Shorter Iso-chains Derived from Alternative Digestion Strategies 2446.2.4.1 SUMO-Isopeptides with Atypical Iso-chains Generated from Tryptic Digestion 2446.2.4.2 Dual Proteolytic Enzyme Digestion with Trypsin and Chymotrypsin 2476.2.4.3 Proteolytic Enzyme and Chemical Digestion with Trypsin and Acid 2486.2.5 MS Analysis of Modified Ub- and SUMO-Isopeptides under CID Conditions 2506.2.6 SPITC Modification 2516.2.7 Dimethyl Modification 2526.2.8 m-TRAQ Modification 2566.3 Enrichment and Global Analysis of Isopeptides 2596.3.1 Overview of Enrichment Approaches 2596.3.2 K-GG Antibody 2606.3.3 COFRADIC 2626.3.4 SUMOylation Enrichment 2636.4 Concluding Remarks and Recommendations 265References 2677 The Deimination of Arginine to Citrulline 275Andrew J. Creese and Helen J. Cooper7.1 Overview of Arginine to Citrulline Conversion: Biological Importance 2757.2 Mass Spectrometry-Based Proteomics 2797.3 Liquid Chromatography and Mass Spectrometry Behavior of Citrullinated Peptides 2837.4 Global Analysis of Citrullination 2887.5 Enrichment Strategies 2917.6 Bioinformatics 2967.7 Concluding Remarks 297Acknowledgements 297References 2978 Glycation of Proteins 307Naila Rabbani and Paul J. Thornalley8.1 Overview of Protein Glycation 3078.2 Mass Spectrometry Behavior of Glycated Peptides 3158.3 Global Analysis of Glycation 3188.4 Enrichment Strategies 3198.5 Bioinformatics 3208.6 Concluding Remarks 323Acknowledgements 324References 3249 Biological Significance and Analysis of Tyrosine Sulfation 333Éva Klement, Éva Hunyadi-Gulyás and Katalin F. Medzihradszky9.1 Overview of Protein Sulfation 3339.2 Mass Spectrometry Behavior of Sulfated Peptides 3349.3 Enrichment Strategies and Global Analysis of Sulfation 3409.4 Sulfation Site Predictions 3429.5 Summary 343Acknowledgements 344References 34410 The Application of Mass Spectrometry for the Characterization of Monoclonal Antibody-Based Therapeutics 351Rosie Upton, Kamila J. Pacholarz, David Firth, Sian Estdale and Perdita E. Barran10.1 Introduction 35110.1.1 Antibody Structure 35210.1.2 N-Linked Glycosylation 35410.1.3 Antibody-Drug Conjugates 35510.1.4 Biosimilars 35610.2 Mass Spectrometry Solutions to Characterizing Monoclonal Antibodies 35810.2.1 Hyphenated Mass Spectrometry (X-MS) Techniques to Study Glycosylation Profiles 35910.2.2 Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) to Characterize Monoclonal Antibody Structure 36110.2.3 Native Mass Spectrometry and the Use of IM-MS to Probe Monoclonal Antibody Structure 36510.3 Advanced Applications 36910.3.1 Quantifying Glycosylation 36910.3.2 Antibody-Drug Conjugates 37010.3.3 Biosimilar Characterization 37210.4 Concluding Remarks 374References 374Index 387