List of Contributors xi
 
Preface xv
 
1 Introduction 1
Rebecca 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 17
Dean 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 89
David 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 ß?-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 161
Caroline Evans
 
4.1 Overview of Protein Acetylation and Methylation 161
 
4.1.1 Protein Acet