Analysis of Protein Post–Translational Modifications by Mass Spectrometry

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.

List of contributors

Preface

Chapter 1: Introduction
Rebecca Pferdehirt, Florian Gnad, Jennie R. Lill

1.1. Post–translational modification of proteins

1.2. Global versus targeted analysis strategies

1.3. Mass spectrometric analysis methods for the detection of post translational modifications

1.4. The importance of bioinformatics

References

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

2.1. Introduction to Protein Phosphorylation

2.2. Analysis of Protein Phosphorylation by Mass Spectrometry

2.3. Global analysis of protein phosphorylation by mass spectrometry

2.4. Sample preparation and enrichment strategies for phosphoprotein analysis by mass spectrometry

2.5. Multidimensional separations for deep coverage of the phosphoproteome

2.6. Computational and bioinformatic tools for phosphoproteomics

2.7. Concluding Remarks

References

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

3.1. Introduction

3.2. General structures of carbohydrates

3.3. Isolation and purification of glycoproteins

3.4. Mass spectrometry of intact glycoproteins

3.5. Site analysis

3.6. Glycan release

3.7. Analysis of released glycans

3.8. Mass spectrometry of glycans

3.9. Computer interpretation of MS data

3.10. Total glycomics methods

3.11. Conclusions

 References

Chapter 4: Protein Acetylation and Methylation
Caroline Evans

4.1. Overview of protein acetylation and methylation

4.2. Mass spectrometry behavior of modified peptides

4.3. Global analysis

4.4. Enrichment

4.5. Bioinformatics

4.6. Summary

References

Chapter 5: Tyrosine Nitration
Xianquan Zhan, Ying Long, Dominic M. Desiderio

5.1. Overview of tyrosine nitration

5.2. Mass spectrometry behavior of nitrated peptides

5.3. Global analysis of tyrosine nitration

5.4. Enrichment strategies

5.5. Concluding remarks

Abbreviations

Acknowledgements

References

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

6.1. Overview of SUMO and Ub

6.2. Mass Spectrometry Behaviour of Isopeptides

6.3. Enrichment and Global Analysis of Isopeptides

6.4. Concluding Remarks and Recommendations

References

Chapter 7: The Deimination of Arginine to Citrulline
Andrew J. Creese, Helen J. Cooper

7.1. Overview of arginine to citrulline conversion: Biological importance

7.2. Mass spectrometry–based proteomics

7.3. Liquid chromatography and mass spectrometry behavior of citrullinated peptides

7.4. Global analysis of citrullination

7.5. Enrichment strategies

7.6. Bioinformatics

7.7. Concluding remarks

Acknowledgements

References

Chapter 8: Glycation of Proteins
Naila Rabbani and Paul J. Thornalley

8.1. Overview of protein glycation

8.2. Mass spectrometry behavior of glycated peptides

8.3. Global analysis of glycation

8.4. Enrichment strategies

8.5. Bioinformatics

8.6. Concluding remarks

References

Chapter 9: Biological Significance and Analysis of Tyrosine Sulfation
Éva Klement 1, Éva Hundyadi–Gulyás 1, Katalin F. Medzihradszky

9.1. Overview of protein sulfation

9.2. Mass spectrometry behavior of sulfated peptides

9.3. Enrichment strategies and global analysis of sulfation

9.4. Sulfation site predictions

References

Chapter 10: The Application of Mass Spectrometry for the Characterisation of Monoclonal Antibody–Based Therapeutics
Rosie Upton, Kamila J. Pacholarz, David Firth, Sian Estdale and Perdita E. Barran

10.1. Introduction

10.2. Antibody Structure

10.3. N–linked Glycosylation

10.4. Antibody–Drug Conjugates

10.5. Biosimilars

10.6. Sorption thermodynamics of heavy metals by ENMs

10.7. Factors influencing heavy metal sorption by ENMs

10.8. Summary and future perspectives

References

Index