_ Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation _ Discussion of the chemistry behind each modification, along with key methods and references _ Contributions from some of the leading researchers in the field _ A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research
_ Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation _ Discussion of the chemistry behind each modification, along with key methods and references _ Contributions from some of the leading researchers in the field _ A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research
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.
Inhaltsangabe
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
