Analysis of Protein Post-Translational Modifications by Mass Spectrometry - John R. Griffiths - ebook

Analysis of Protein Post-Translational Modifications by Mass Spectrometry ebook

John R. Griffiths

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* 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

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Table of Contents

Cover

Title Page

Copyright

List of Contributors

Preface

Chapter 1: Introduction

1.1 Post-translational Modification of Proteins

1.2 Global versus Targeted Analysis Strategies

1.3 Mass Spectrometric Analysis Methods for the Detection of PTMs

1.4 The Importance of Bioinformatics

Acknowledgements

References

Chapter 2: Identification and Analysis of Protein Phosphorylation by Mass Spectrometry

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 Bioinformatics Tools for Phosphoproteomics

2.7 Concluding Remarks

References

Chapter 3: Analysis of Protein Glycosylation by Mass Spectrometry

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

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

5.1 Overview of Tyrosine Nitration

5.2 MS Behavior of Nitrated Peptides

5.3 Global Analysis of Tyrosine Nitration

5.4 Enrichment Strategies

5.5 Concluding Remarks

Acknowledgements

References

Chapter 6: Mass Spectrometry Methods for the Analysis of Isopeptides Generated from Mammalian Protein Ubiquitination and SUMOylation

6.1 Overview of Ub and SUMO

6.2 Mass Spectrometry Behavior 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

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

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

Acknowledgements

References

Chapter 9: Biological Significance and Analysis of Tyrosine Sulfation

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

9.5 Summary

Acknowledgement

References

Chapter 10: The Application of Mass Spectrometry for the Characterization of Monoclonal Antibody-Based Therapeutics

10.1 Introduction

10.2 Mass Spectrometry Solutions to Characterizing Monoclonal Antibodies

10.3 Advanced Applications

10.4 Concluding Remarks

References

Index

End User License Agreement

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Guide

Cover

Table of Contents

Preface

Begin Reading

List of Tables

Chapter 2: Identification and Analysis of Protein Phosphorylation by Mass Spectrometry

Table 2.1 Site localization tools.

Chapter 3: Analysis of Protein Glycosylation by Mass Spectrometry

Table 3.1 Structures of the cores of O-glycans 27.

Table 3.2 Matrices for MALDI mass spectrometry of carbohydrates.

Table 3.3 Residue masses of common monosaccharides.

Table 3.4 Ions defining structural features in the negative ion spectra of N-linked glycans.

Chapter 8: Glycation of Proteins

Table 8.1 Components of the fructosamine proteome.

Table 8.2 Components of the dicarbonyl proteome.

Chapter 9: Biological Significance and Analysis of Tyrosine Sulfation

Table 9.1 Reliably assigned human sulfo-Tyr sites from UniProt May 2015.

Table 9.2 Prediction softwares tested with a few proteins with reliable site assignments.

Chapter 10: The Application of Mass Spectrometry for the Characterization of Monoclonal Antibody-Based Therapeutics

Table 10.1 Averaged relative MS responses for the quantification of nonglycosylated, afucosylated, and fucosylated Fc/2 subunits of EU and US Herceptin® and a trastuzumab sample following digestion with IdeS, EndoS, and EndoS2.

WILEY SERIES ON MASS SPECTROMETRY

 

Series Editors

Dominic M. Desiderio

Departments of Neurology and Biochemistry University of Tennessee Health Science Center

 

Joseph A. Loo

Department of Chemistry and Biochemistry UCLA

 

Founding Editors

 

Nico M. M. Nibbering (1938-2014)

Dominic M. Desiderio

 

A complete list of the titles in this series appears at the end of this volume.

Analysis of Protein Post-Translational Modifications by Mass Spectrometry

 

Edited by John R. Griffiths and Richard D. Unwin

 

 

 

Copyright © 2017 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permissions.

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Library of Congress Cataloging-in-Publication Data:

Names: Griffiths, John R., 1964- editor. | Unwin, Richard D., editor.

Title: Analysis of Protein Post-Translational Modifications by Mass Spectrometry / edited by John R. Griffiths, Richard D. Unwin.

Description: Hoboken, New Jersey : John Wiley & Sons, 2016. | Includes bibliographical references and index.

Identifiers: LCCN 2016024928 | ISBN 9781119045854 (cloth) | ISBN 9781119250890 (epub)

Subjects: LCSH: Post-translational modification. | Mass spectrometry.

Classification: LCC QH450.6.A53 2016 | DDC 572/.645-dc23

LC record available at https://lccn.loc.gov/2016024928

List of Contributors

Roland S. Annan

Proteomics and Biological Mass Spectrometry Laboratory

GlaxoSmithKline

Collegeville, PA

USA

 

Perdita E. Barran

Manchester Institute of Biotechnology

The University of Manchester

Manchester

UK

 

Navin Chicooree

Cancer Research UK Manchester Institute

The University of Manchester

Manchester, UK

 

Helen J. Cooper

School of Biosciences

University of Birmingham

Birmingham, UK

 

Andrew J. Creese

School of Biosciences

University of Birmingham

Birmingham, UK

 

Dominic M. Desiderio

The Charles B. Stout Neuroscience Mass Spectrometry Laboratory Department of Neurology

University of Tennessee Health Science Center

Memphis, TN

USA

 

Sian Estdale

Covance Laboratories

Harrogate

UK

 

Caroline A. Evans

Department of Chemical and Biological Engineering

University of Sheffield

Sheffield, UK

 

David Firth

Covance Laboratories Ltd.

Harrogate, UK

 

Florian Gnad

Proteomics and Biological Resources

Genentech Inc

South San Francisco, CA

USA

 

John Griffiths

Cancer Research UK Manchester Institute

The University of Manchester

Manchester

UK

 

David J. Harvey

Department of Biochemistry

University of Oxford

Oxford

UK

 

Éva Hunyadi-Gulyás

Institute of Biochemistry

Biological Research Centre of the Hungarian Academy of Sciences

Szeged

Hungary

 

Éva Klement

Institute of Biochemistry

Biological Research Centre of the Hungarian Academy of Sciences

Szeged

Hungary

 

Jennie R Lill

Proteomics and Biological Resources

Genentech Inc

South San Francisco, CA

USA

 

Ying Long

Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital

Central South University

Changsha, Hunan

R. China

 

Dean E. McNulty

Proteomics and Biological Mass Spectrometry Laboratory

GlaxoSmithKline

Collegeville, PA

USA

 

Katalin F. Medzihradszky

Department of Pharmaceutical Chemistry

University of California San Francisco

San Francisco, CA

USA

 

Kamila J. Pacholarz

Manchester Institute of Biotechnology

The University of Manchester

Manchester

UK

 

Rebecca Pferdehirt

Proteomics and Biological Resources

Genentech Inc

South San Francisco, CA

USA

 

Naila Rabbani

Warwick Systems Biology Centre

University of Warwick

Coventry

UK

 

Timothy W. Sikorski

Proteomics and Biological Mass Spectrometry Laboratory

GlaxoSmithKline

Collegeville, PA

USA

 

Duncan L. Smith

Cancer Research UK Manchester Institute

The University of Manchester

Manchester

UK

 

Paul J. Thornalley

Warwick Medical School, Clinical Sciences Research Laboratories

University of Warwick

Coventry

UK

 

Richard Unwin

Centre for Advanced Discovery and Experimental Therapeutics (CADET)

Central Manchester University Hospitals NHS Foundation Trust

Manchester

UK

 

Rosie Upton

Manchester Institute of Biotechnology

The University of Manchester

Manchester

UK

 

Xianquan Zhan

Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital

Central South University

Changsha, Hunan

R. China

Preface

While preparing a recent review article in Mass Spectrometry Reviews on the analysis of post-translational modifications (PTMs) by mass spectrometry, we realized that, although there is much excellent work and many new tools being developed in this area, the field was lacking a coherent resource where these advances could be easily and readily accessed both by experts and those wishing to begin such studies. We subsequently decided that there was a need for a more comprehensive description of some of these modifications, and their analysis by mass spectrometry, in the form of a textbook. Since a detailed discussion of multiple modifications was required, it rapidly became apparent that this would require the support of experts in their own specialized fields. We are, therefore, grateful that a number of mass spectrometrists from around the world whom we, and others involved in proteomics, consider to be experts in the analysis of specific PTMs, agreed to contribute to this effort.

The aim of the book is to provide the reader with an understanding of the importance of the protein modifications under discussion in a biological context, and to yield insights into the analytical strategies, both in terms of sample preparation, chemistry, and analytical considerations required for the mass spectrometric determination of the presence, location, and function of selected important PTMs.

The scene is ably set with a concise introduction to the general strategies employed in PTM analysis by mass spectrometry, covering some of the key technologies which are referred to in more detail in subsequent chapters. Of course, well-known and more thoroughly investigated modifications such as phosphorylation, glycosylation, and acetylation are described in this work in great detail. However, other PTMs are garnering interest within the field and play major roles in protein function both in normal cellular regulation and in the disease setting. These PTMs are generally less well studied to date, and include, for example, tyrosine sulfation, glycation, nitration, and citrullination - the conversion of arginine to citrulline. The analysis of ubiquitination and SUMOylation, both of which involve the addition of a second, small protein to the target in a complex regulation of protein localization, activity, and stability completes the array of modifications included in this book. In addition, the book rounds off with a description of one of the current “hot topics” in mass spectrometry: that of top-down studies of intact protein structure and modification, using the example of the characterization of monoclonal antibodies.

As editors, it has been our joint pleasure and privilege to have been given the opportunity to read at first hand these works and to compile them into a book of which we are very proud. On behalf of both of us we would like to express our sincere thanks and appreciation for the hard work and generosity given by all of the contributors.

Finally, to you the reader, we hope that you are able to use this book in your research, either as a reference book to dip into from time to time, to introduce you to new methodologies or new ideas to help support your work, or as a means of gaining a greater understanding of the analysis of PTMs by mass spectrometry from some expert scientists.

April 2016

John R. GriffithsRichard D. UnwinManchester, UK

Chapter 1Introduction

Rebecca Pferdehirt, Florian Gnad and Jennie R. Lill

Proteomics and Biological Resources, Genentech Inc., South San Francisco, CA, USA

1.1 Post-translational Modification of Proteins

While the human proteome is encoded by approximately 20,000 genes [1, 2], the functional diversity of the proteome is orders of magnitude larger because of added complexities such as genomic recombination, alternative transcript splicing, or post-translational modifications (PTMs) [3, 4]. PTMs include the proteolytic processing of a protein or the covalent attachment of a chemical or proteinaceous moiety to a protein allowing greater structural and regulatory diversity. Importantly, PTMs allow for rapid modification of a protein in response to a stimulus, resulting in functional flexibility on a timescale that traditional transcription and translation responses could never accommodate. PTMs range from global modifications such as phosphorylation, methylation, ubiquitination, and glycosylation, which are found in all eukaryotic species in all organs, to more specific modifications such as crotonylation (thought to be spermatozoa specific) and hypusinylation (specific for EIF5a), which govern more tight regulation of associated proteins. Taken together, over 200 different types of PTMs have been described [5], resulting in an incredibly complex repertoire of modified proteins throughout the cell.

The addition and subtraction of PTMs are controlled by tight enzymatic regulation. For example, many proteins are covalently modified by the addition of a phosphate group onto tyrosine, serine, or threonine residues in a process called phosphorylation [6]. Phosphorylation is catalyzed by a diverse class of enzymes called kinases [7], whereas these phosphomoieties are removed by a second class of enzymes referred to as phosphatases. The tight regulation of kinases and phosphatases often creates “on/off” switches essential for regulation of sensitive signaling cascades. There are some exceptions to this rule however, and the hunt is still underway for the ever-elusive hypusine [8] removing enzyme or putative enzymes responsible for the removal of protein arginine methylation. However, it is also possible that proteins bearing these PTMs are modulated or removed from the cell by other mechanisms of action. For example, proteolysis is rarely (if ever) reversible, and many proteins (e.g., blood clotting factors and digestive enzymes) are tightly governed by irreversible cleavage events where the active form is created after proteolysis of a proenzyme.

While PTMs such as phosphorylation and lysine acetylation exist in a binary “on/off” state, many other PTMs exhibit much more complex possible modification patterns. For example, lysine residues can be modified by covalent attachment of the small protein ubiquitin, either by addition of a single ubiquitin or by addition of ubiquitin polymers. In the latter case ubiquitin itself is used as the point of attachment for addition of subsequent ubiquitin monomers [9]. To add another layer of complexity, ubiquitin has seven lysines (K6, K11, K27, K29, K33, K48, and K63), each of which may be used as the point of polyubiquitin chain linkage, and each of which has a different functional consequence. For example, K63-linked chains are associated with lysosomal targeting, whereas K48-linked chains trigger substrate degradation by the proteasome. Thus, even within one type of PTM, multiple subtypes exist, further expanding the functional possibilities of protein modification.

In addition, many proteins are modified on multiple residues by different types of PTMs. A classic example is the PTM of histones. Histones are nuclear proteins that package and compact eukaryotic DNA into structural units called nucleosomes, which are the basic building blocks of chromatin and essential for regulation of gene expression. The C-termini of histones are composed of unstructured tails that protrude from nucleosomes and are heavily modified by methylation, acetylation, ubiquitylation, phosphorylation, SUMOylation, and other PTMs [10]. Overall, 26 modified residues on a single-core histone have been identified, and many of these residues can harbor multiple PTM types. In a generally accepted theory referred to as the “histone code,” the combination of PTMs on all histones comprising a single nucleosome or group of nucleosomes regulates fine-tuned expression of nearby genes.

As we begin to uncover the modified proteome, the importance of the interplay between multiple different PTMs has become increasingly apparent. One classic example is the involvement of both protein phosphorylation and ubiquitylation in the regulation of signaling networks [11]. Protein phosphorylation commonly promotes subsequent ubiquitylation, and the activities of ubiquitin ligases are also frequently regulated through phosphorylation. In a recent study by Ordureau et al., quantitative proteomic studies were employed to describe the PINK1 kinase–PARKIN UB ligase pathway and its disruption in Parkinson’s disease [12]. The authors describe a feedforward mechanism where phosphorylation of PARKIN by PINK1 occurs upon mitochondrial damage, leading to ubiquitylation of mitochondria and mitochondrial proteins by PARKIN. These newly formed ubiquitin chains are then themselves phosphorylated by PINK1, which promotes association of phosphorylated PARKIN with polyubiquitin chains on the mitochondria, and ultimately results in signal amplification. This model exemplifies how intricate interactions between multiple different PTMs regulate protein localization, interactions, activity, and ultimately essential cellular processes.

Recent advances in mass spectrometry methods, instrumentation, and bioinformatics analyses have enabled the identification and quantification of proteome-wide PTMs. For example, it is now a common practice to identify ten thousand phosphorylation sites in a single phosphoproteome enrichment experiment [13]. In addition, precise quantitation allows a deeper understanding of the combinations and occupancy of PTMs within a given protein. Such MS-based PTM analyses have led to previously impossible discoveries, advancing our understanding of the role of PTMs in diverse biological processes.

1.2 Global versus Targeted Analysis Strategies

Detection of PTMs by mass spectrometry can be achieved via global or targeted methods. The biological pathway of interest usually determines the type of PTM to be analyzed and associated methods. In a more targeted approach, researchers decide to investigate PTMs, because a protein of interest shows a higher than expected molecular weight or multiple bands by western blot after application of a stimulus, thus prompting speculation as to whether this could be due to PTM. Either way, the first step in PTM mapping is to determine the type of PTM of interest. In some cases the observed mass shift in a mass spectrometer indicates a certain PTM type. Many PTMs, however, result in the same mass addition (e.g., +42 Da for both acetylation and trimethylation). One powerful strategy in determining PTM identity involves the employment of the enzymes responsible for PTM removal. For example, after antibody enrichment of a modified protein, the antibody-bound protein can be incubated with general phosphatases, deubiquitinating enzymes (DUBs), or deSUMOylating enzymes (SENPs), and PTM removal can be assayed by western blot. Another method for PTM identification is western blotting with PTM specific “pan-antibodies.” Many commercially available antibodies exist for this purpose, recognizing common PTMs such as acetylation, methylation, ubiquitylation, and phosphorylation or even more rare PTMs such as crotonyl-, malonyl- or glutaryl-lysine modification. Once the type of PTM that is decorating a protein has been identified, the next step is to attempt to map the amino acid residue(s) that bear this modification.

One of the first applications of mass spectrometry in protein research was the mapping of a PTM on a single protein [14]. A commonly used approach involves protein-level immunoprecipitation followed by separating the captured proteins by SDS-PAGE, excising the higher molecular weight band, and performing in-gel tryptic digestion followed by LC-MS/MS. By searching for mass shifts indicative of the suspected modification(s), PTM-containing peptides can be identified and the PTM site mapped back to the protein. The strategy of identifying proteins in complex mixtures by digesting them into peptides, sequencing the resulting peptides by tandem mass spectrometry (MS/MS), and determining peptide and protein identity through automated database searching is referred to as shotgun proteomics and is one of the most popular analysis strategies in proteomics [15]. This protein-level enrichment approach, however, is dependent on sufficient levels of the modified protein compared to unmodified and the availability of protein-specific antibodies for immunoprecipitation. It is also possible that modifications may occur within the antibody epitope, blocking enrichment of the modified form altogether.

Researchers are commonly interested in analyzing PTMs from a complex mixture of proteins rather than on only one substrate. This can be a challenge, since modified peptides often occur in substoichiometric levels compared to unmodified versions and also may ionize less efficiently by electrospray ionization (ESI). However, several enrichment strategies exist, allowing for reduction of sample complexity and easier detection of the modified peptide species. Peptide-level immunoprecipitation using antibodies specific to a given PTM is an increasingly popular method of enrichment prior to MS. While this strategy can be employed for any PTM enrichment, it has been most commonly used for mapping ubiquitination sites. Tryptic digestion of ubiquitinated proteins generates a diglycine remnant attached to the ubiquitinated lysine residue (K-GG) that can be recognized by antibodies. The resulting mass shift of +114.0429 Da can be detected by MS/MS. Not only has K-GG peptide immunoaffinity enrichment enabled the identification of hundreds of ubiquitination sites on a global level but it has also been shown to enhance identification of ubiquitination sites on individual proteins, when compared to protein-level IP coupled with MS/MS [16].

To understand the biological significance of a specific PTM, it is also important to determine the PTM site occupancy or percentage of a protein’s total population that is modified. Quantification of site occupancy can be accomplished by combining antibody peptide enrichment with stable isotope-labeled internal standards of the same sequence, a method termed stable isotope standards and capture by anti-peptide antibodies (SISCAPA) [17]. By coupling immunoprecipitation with stable isotope dilution multiple reaction monitoring (SID-MRM), absolute quantitation of both modified and unmodified protein populations can be determined in a high-throughput, multiplexing-compatible fashion [18].

In addition to antibody-based enrichment approaches, several strategies for chemical enrichment of PTM-containing subproteomes have been developed. These approaches can also be coupled with the use of stable isotope standard peptides and SRM/MRM for accurate quantification of PTM dynamics. The most widely studied PTM, with the most variety of enrichment methods available, is phosphorylation. Global analysis of serine, threonine, and tyrosine phosphorylation can be achieved by a combination of peptide fractionation using strong cation exchange (SCX) followed by further enrichment with immobilized metal affinity chromatography (IMAC). The SCX/IMAC approach allows for enrichment of phosphorylated peptides to over 75% purity and ultimately identification of over 10,000 phosphorylation sites from 5 mg of starting protein [13, 19]. Another common approach for selective enrichment of the phosphoproteome is using metal oxide affinity chromatography (MOAC) such as titanium dioxide (TiO2) [20] or aluminum hydroxide (Al(OH3)) [21]. MOAC methods have been reported to achieve higher sensitivity than IMAC (at the cost of lower specificity though). The combination of multiple enrichment approaches may ultimately be the best approach.

Phosphopeptide enrichment strategies can also be applied on crude protein extract to enrich for entire phosphoproteins. Enriched fractions are typically separated by two-dimensional gel electrophoresis (2D-GE) or sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). In either case, each observed protein spot/band is quantified by its staining intensity, and selected spots/bands are excised, digested, and analyzed by MS. The advantage of phosphoprotein enrichment is that intact proteins are separated, and the molecular weight and isoelectric point of proteins can be determined. This greatly aids in protein identification by MS. However, protein-level enrichment has several disadvantages, including loss of small or hydrophobic proteins during precipitation steps, less specific enrichment when compared to phosphopeptides, and difficulty in identifying low-abundance proteins or modifications [22].

In summary, both targeted and global methods for PTM identification have been significantly tuned in recent years but are still facing challenges. The choice of method is usually dictated by the biological question. However, global strategies are becoming increasingly popular due to their versatility, sensitivity, and ability to collect a wealth of data, triggering new hypotheses that ask for validation by targeted experiments.

1.3 Mass Spectrometric Analysis Methods for the Detection of PTMs

Mass spectrometers are powerful, analytical tools that have evolved rapidly over the past few decades to become the instrument of choice for protein and peptide characterization. Mass spectrometry is often used in parallel to other techniques such as western blot analysis or protein microarrays for detecting and quantifying PTMs. One of the main advantages of mass spectrometry is the ability to rapidly analyze many samples in a high-throughput manner. Mass spectrometric analyses can be divided into three main strategies: “bottom-up,” “middle-down,” and “top-down” proteomic approaches [23]. Laboratories typically employ bottom-up proteomic methodologies to characterize PTMs. Proteins of interest are purified and proteolytically digested with an enzyme such as trypsin, with resultant peptides being separated by reversed-phase chromatography or another analytical method compatible with mass spectrometric analysis. One of several fragmentation methods and ion detection methodologies can then be employed (see Sections 1.3.1–1.3.4 for description of the various types of bottom-up proteomic analyses). It is common to associate “data-dependent” MS/MS analysis with bottom-up approaches, where resulting peptide spectra are then pieced back together in silico to give an overview of the protein and its PTMs.

In top-down proteomics, intact protein ions or large protein fragments are subjected to gas-phase fragmentation for MS analysis. Here, a variety of fragmentation mechanisms can be employed to induce dissociation and mass spectrometric analysis of the protein including collision-induced dissociation (CID), electron transfer dissociation (ETD), and electron capture dissociation (ECD) [24–26]. High-resolution mass detectors such as the quadrupole–time of flight (Q-TOF), Fourier transform ion cyclotron resonance (FT-ICR), or orbitrap mass spectrometers are typically employed as the spectra generated from top-down fragmentation tend to be highly charged and therefore difficult to resolve without high-resolution power. Top-down proteomics to date has been a less popular tool for characterizing PTMs than bottom-up analysis. However, it is an invaluable tool in cases where a bottom-up approach would lose contextual information about combinatorial PTM distribution (e.g., in the case of histone PTM analysis [27]). The middle-down approach has more commonly been employed as a strategy whereby a proteolytic enzyme can be used to generate longer polypeptides from a protein of interest and has shown utility in analyzing complex PTMs such as the histone code [28, 29]. Compared to middle-down and top-down methods, the bottom-up approach often offers better front-end separation of peptides, typically equating to higher sensitivity and selectivity. There are however some limitations to the bottom-up approach including the risk of low sequence coverage, particularly when employing a single proteolytic enzyme such as trypsin where cleavage may result in peptides yielding chemophysical properties with poor analytical attributes, such as size or substandard hydrophobicity.

1.3.1 Data-Dependent and Data-Independent Analyses

The type of mass spectrometric analysis performed for PTM detection depends on whether a single protein with a single PTM is being analyzed or if it is a global approach, such as a global phosphoproteomic analysis. When targeting a single protein or a subset of proteins for a PTM of interest, a straightforward strategy is to perform an enzymatic digestion followed by data-dependent MS/MS analysis of peptides. In this approach, the intact molecular weight of each peptide in the full MS scan is analyzed, and then a selection of the most abundant peptides in the full MS scan are sequentially selected for fragmentation using one of several fragmentation methods. The resulting spectra are then analyzed either through de novo sequencing or more commonly using a search algorithm such as SEQUEST [30], Mascot [31], or Andromeda [32]. Peptides are then scored using an algorithm to calculate the false discovery rate or validated through manual spectral interpretation or by incorporation of a synthetic standard.

In traditional data-dependent acquisition (DDA), a proteomic sample is digested into peptides, separated often by reversed-phase chromatography, and ionized and analyzed by mass spectrometry. Typically instruments are programmed to select any ions that fall above a certain intensity threshold in full MS for subsequent MS/MS fragmentation. Although a powerful and highly utilized technique, the method is indeed biased to peptides that are of higher abundance, and lower level moieties such as post-translationally modified peptides may go undetected using DDA. Several years ago an alternative methodology called data-independent acquisition (DIA) was introduced which has slowly been gaining momentum [33]. In DIA analysis, all peptides within a defined mass-to-charge (m/z) window are subjected to fragmentation; the analysis is repeated as the mass spectrometer walks along the full m/z range. This results in the identification of lower level peptides, for example, post-translationally modified species present at substoichiometric levels compared to their nonmodified counterparts. It also allows accurate peptide quantification without being limited to profiling predefined peptides of interest and has proved useful in the biomarker community where quantitation on complex samples is routinely employed. The DIA method has matured in terms of utility over the past few years with the introduction of more user friendly and accurate search algorithms and spectral library search capabilities [34, 35]. Its utility as a tool to identify complex, low level, and isobaric amino acids has also recently been reported [36, 37].

1.3.2 Targeted Analyses

In addition to data-dependent approaches, targeted methods also exist whereby specific ion transitions can be monitored. These various targeted approaches are summarized in Figure 1.1