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Analysis of Protein Post–Translational Modifications by Mass Spectrometry. Wiley Series on Mass Spectrometry

  • ID: 3736376
  • Book
  • 416 Pages
  • John Wiley and Sons Ltd
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Provides a comprehensive overview of the various ways mass spectrometry can be used for the detection and analysis of protein post–translational modifications

Post–translational modification of a protein adds significant diversity and function to the proteome. It may involve the addition of functional groups to amino acids, covalent linkage of other proteins or fatty acids, a change in the chemical nature of an amino acid or structural changes resulting from proteolytic cleavage or disulfide bridge formation. Over the last 10 years, mass spectrometry has become a critical tool in the analysis of proteins structure, quantity and modification. Of the 300 or so PTMs known, this book sets out to describe mass spectrometry–based methods for the analysis of some of the most well studied.

Providing a single starting reference point for this growing and important field, with contributions from renowned experts in the analysis of all of the major PTMs and containing key background, references and summarizing the current state–of–the–art, this book is an indispensable tool for students and practitioners of protein 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 xi

Preface xv

1 Introduction 1Rebecca Pferdehirt, Florian Gnad and Jennie R. Lill

1.1 Post–translational Modification of Proteins 1

1.2 Global versus Targeted Analysis Strategies 3

1.3 Mass Spectrometric Analysis Methods for the Detection of PTMs 5

1.3.1 Data –Dependent and Data –Independent Analyses 6

1.3.2 Targeted Analyses 7

1.3.3 Multiple Reaction Monitoring 8

1.3.4 Multiple Reaction Monitoring Initiated Detection and Sequencing 9

1.4 The Importance of Bioinformatics 9

Acknowledgements 11

References 11

2 Identification and Analysis of Protein Phosphorylation by Mass Spectrometry 17Dean E. McNulty, Timothy W. Sikorski and Roland S. Annan

2.1 Introduction to Protein Phosphorylation 17

2.2 Analysis of Protein Phosphorylation by Mass Spectrometry 25

2.3 Global Analysis of Protein Phosphorylation by Mass Spectrometry 39

2.4 Sample Preparation and Enrichment Strategies for Phosphoprotein Analysis by Mass Spectrometry 46

2.5 Multidimensional Separations for Deep Coverage of the Phosphoproteome 54

2.6 Computational and Bioinformatics Tools for Phosphoproteomics 57

2.7 Concluding Remarks 65

References 66

3 Analysis of Protein Glycosylation by Mass Spectrometry 89David J. Harvey

3.1 Introduction 89

3.2 General Structures of Carbohydrates 89

3.2.1 Protein –Linked Glycans 90

3.3 Isolation and Purification of Glycoproteins 94

3.3.1 Lectin Affinity Chromatography 95

3.3.2 Boronate –Based Compounds 95

3.3.3 Hydrazide Enrichment 96

3.3.4 Titanium Dioxide Enrichment of Sialylated Glycoproteins 96

3.4 Mass Spectrometry of Intact Glycoproteins 96

3.5 Site Analysis 96

3.6 Glycan Release 98

3.6.1 Use of Hydrazine 99

3.6.2 Use of Reductive –Elimination 99

3.6.3 Use of Enzymes 100

3.7 Analysis of Released Glycans 102

3.7.1 Cleanup of Glycan Samples 102

3.7.2 Derivatization 102

3.7.2.1 Derivatization at the Reducing Terminus 102

3.7.2.2 Derivatization of Hydroxyl Groups: Permethylation 104

3.7.2.3 Derivatization of Sialic Acids 106

3.7.3 Exoglycosidase Digestions 106

3.7.4 HPLC and ESI 107

3.8 Mass Spectrometry of Glycans 107

3.8.1 Aspects of Ionization for Mass Spectrometry Specific to the Analysis of Glycans 107

3.8.1.1 Electron Impact (EI) 107

3.8.1.2 Fast Atom Bombardment (FAB) 108

3.8.1.3 Matrix –Assisted Laser Desorption/Ionization (MALDI) 108

3.8.1.4 Electrospray Ionization (ESI) 113

3.8.2 Glycan Composition by Mass Spectrometry 114

3.8.3 Fragmentation 114

3.8.3.1 Nomenclature of Fragment Ions 116

3.8.3.2 In –Source Decay (ISD) Ions 116

3.8.3.3 Postsource Decay (PSD) Ions 117

3.8.3.4 Collision –Induced Dissociation (CID) 117

3.8.3.5 Electron Transfer Dissociation (ETD) 118

3.8.3.6 Infrared Multiphoton Dissociation (IRMPD) 118

3.8.3.7 MSn 118

3.8.3.8 Fragmentation Modes of Different Ion Types 119

3.8.4 Ion Mobility 126

3.8.5 Quantitative Measurements 128

3.9 Computer Interpretation of MS Data 128

3.10 Total Glycomics Methods 130

3.11 Conclusions 131

Abbreviations 131

References 133

4 Protein Acetylation and Methylation 161Caroline Evans

4.1 Overview of Protein Acetylation and Methylation 161

4.1.1 Protein Acetylation 161

4.1.2 Protein Methylation 162

4.1.3 Functional Aspects 163

4.1.4 Mass Spectrometry Analysis 163

4.2 Mass Spectrometry Behavior of Modified Peptides 164

4.2.1 MS Fragmentation Modes 164

4.2.2 Acetylation – and Methylation –Specific Diagnostic Ions in MS Analysis 165

4.2.3 Application of MS Methodologies for the Analysis of PTM Status 168

4.2.4 Quantification Strategies 169

4.2.4.1 Single Reaction Monitoring/Multiple Reaction Monitoring 170

4.2.4.2 Parallel Reaction Monitoring 171

4.2.4.3 Data –Independent Acquisition MS 172

4.2.4.4 Ion Mobility MS 173

4.2.5 Use of Stable Isotope Labeled Precursors 174

4.2.5.1 Dynamics of Acetylation and Methylation 174

4.2.5.2 Stoichiometry of Acetylation and Methylation 175

4.3 Global Analysis 176

4.3.1 Top –Down Proteomics 176

4.3.2 Middle Down 177

4.4 Enrichment 178

4.4.1 Immunoaffinity Enrichment 178

4.4.2 Reader Domain –Based Capture 179

4.4.2.1 Kac –Specific Capture Reagents 179

4.4.2.2 Methyl –Specific Capture Reagents 180

4.4.3 Biotin Switch –Based Capture 180

4.4.4 Enrichment of N –Terminally Acetylated Peptides 181

4.5 Bioinformatics 181

4.5.1 Assigning Acetylation and Methylation Status 182

4.5.2 PTM Repositories and Data Mining Tools 183

4.5.3 Computational Prediction Tools for Acetylation and Methylation Sites 183

4.5.4 Information for Design of Follow –Up Experiments 185

4.6 Summary 185

References 185

5 Tyrosine Nitration 197Xianquan Zhan, Ying Long and Dominic M. Desiderio

5.1 Overview of Tyrosine Nitration 197

5.2 MS Behavior of Nitrated Peptides 199

5.3 Global Analysis of Tyrosine Nitration 208

5.4 Enrichment Strategies 214

5.5 Concluding Remarks 221

Acknowledgements 222

Abbreviations 222

References 223

6 Mass Spectrometry Methods for the Analysis of Isopeptides Generated from Mammalian Protein Ubiquitination and SUMOylation 235Navin Chicooree and Duncan L. Smith

6.1 Overview of Ub and SUMO 235

6.1.1 Biological Overview of Ubiquitin –Like Proteins 235

6.1.2 Biological Overview of Ub and SUMO 236

6.1.3 Biological Functions of Ub and SUMO 236

6.2 Mass Spectrometry Behavior of Isopeptides 237

6.2.1 Terminology of a Ub/Ubl isopeptide 237

6.2.2 Mass Spectrometry Analysis of SUMO –Isopeptides Derived from Proteolytic Digestion 238

6.2.3 Analysis of SUMO –Isopeptides with Typical Full –Length Tryptic Iso –chains 238

6.2.4 Analysis of SUMO –Isopeptides with Atypical Tryptic Iso –chains and Shorter Iso –chains Derived from Alternative Digestion Strategies 244

6.2.4.1 SUMO –Isopeptides with Atypical Iso –chains Generated from Tryptic Digestion 244

6.2.4.2 Dual Proteolytic Enzyme Digestion with Trypsin and Chymotrypsin 247

6.2.4.3 Proteolytic Enzyme and Chemical Digestion with Trypsin and Acid 248

6.2.5 MS Analysis of Modified Ub – and SUMO –Isopeptides under CID Conditions 250

6.2.6 SPITC Modification 251

6.2.7 Dimethyl Modification 252

6.2.8 m –TRAQ Modification 256

6.3 Enrichment and Global Analysis of Isopeptides 259

6.3.1 Overview of Enrichment Approaches 259

6.3.2 K –GG Antibody 260

6.3.3 COFRADIC 262

6.3.4 SUMOylation Enrichment 263

6.4 Concluding Remarks and Recommendations 265

References 267

7 The Deimination of Arginine to Citrulline 275Andrew J. Creese and Helen J. Cooper

7.1 Overview of Arginine to Citrulline Conversion: Biological Importance 275

7.2 Mass Spectrometry –Based Proteomics 279

7.3 Liquid Chromatography and Mass Spectrometry Behavior of Citrullinated Peptides 283

7.4 Global Analysis of Citrullination 288

7.5 Enrichment Strategies 291

7.6 Bioinformatics 296

7.7 Concluding Remarks 297

Acknowledgements 297

References 297

8 Glycation of Proteins 307Naila Rabbani and Paul J. Thornalley

8.1 Overview of Protein Glycation 307

8.2 Mass Spectrometry Behavior of Glycated Peptides 315

8.3 Global Analysis of Glycation 318

8.4 Enrichment Strategies 319

8.5 Bioinformatics 320

8.6 Concluding Remarks 323

Acknowledgements 324

References 324

9 Biological Significance and Analysis of Tyrosine Sulfation 333Éva Klement, Éva Hunyadi–Gulyás and Katalin F. Medzihradszky

9.1 Overview of Protein Sulfation 333

9.2 Mass Spectrometry Behavior of Sulfated Peptides 334

9.3 Enrichment Strategies and Global Analysis of Sulfation 340

9.4 Sulfation Site Predictions 342

9.5 Summary 343

Acknowledgements 344

References 344

10 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. Barran

10.1 Introduction 351

10.1.1 Antibody Structure 352

10.1.2 N–Linked Glycosylation 354

10.1.3 Antibody–Drug Conjugates 355

10.1.4 Biosimilars 356

10.2 Mass Spectrometry Solutions to Characterizing Monoclonal Antibodies 358

10.2.1 Hyphenated Mass Spectrometry (X–MS) Techniques to Study Glycosylation Profiles 359

10.2.2 Hydrogen/Deuterium Exchange Mass Spectrometry (HDX–MS) to Characterize Monoclonal Antibody Structure 361

10.2.3 Native Mass Spectrometry and the Use of IM–MS to Probe Monoclonal Antibody Structure 365

10.3 Advanced Applications 369

10.3.1 Quantifying Glycosylation 369

10.3.2 Antibody–Drug Conjugates 370

10.3.3 Biosimilar Characterization 372

10.4 Concluding Remarks 374

References 374

Index 387

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John R. Griffiths
Richard D. Unwin
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