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Mass Spectrometry-Based Detection and Assignment of Protein Posttranslational Modifications † ‡ † Sophia Doll , and Alma L

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Mass Spectrometry-Based Detection and Assignment of Protein Posttranslational Modifications † ‡ † Sophia Doll , and Alma L This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Reviews pubs.acs.org/acschemicalbiology Mass Spectrometry-Based Detection and Assignment of Protein Posttranslational Modifications † ‡ † Sophia Doll , and Alma L. Burlingame*, † Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517, United States ‡ Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, 82152, Germany ABSTRACT: Recent advances in mass spectrometry (MS)-based proteomics allow the identification and quantitation of thousands of posttranslational modification (PTM) sites in a single experiment. This follows from the development of more effective class enrichment strategies, new high performance instrumentation and bioinformatic algorithms with rigorous scoring strategies. More widespread use of these combined capabilities have led to a vast expansion in our knowledge of the complexity of biological processes mediated by PTMs. The classes most actively pursued include phosphorylation, ubiquitination, O-GlcNAcylation, methylation, and acetylation. Very recently succinylation, SUMOylation, and citrullination have emerged. Among the some 260 000 PTM sites that have been identified in the human proteome thus far, only a few have been assigned to key regulatory and/or other biological roles. Here, we provide an update of MS-based PTM analyses, with a focus on current enrichment strategies coupled with revolutionary advances in high performance MS. Furthermore, we discuss examples of the discovery of recently described biological roles of PTMs and address the challenges of defining site-specific functions. he human genome project revealed only approximately target discovery and validation, and the eventual treatment and T 20 000 protein-coding genes.1 The proteome, however, is prevention of diseases. far more complex and diverse because of post-translational A comprehensive treatment of our earlier level of knowledge modifications (PTMs) and to some extent isoform variations.2 of over 300 types of PTMs, which are known to occur 4 While RNA sequencing detects the expression and sequence physiologically, can be found in the Walsh monograph. Since variations of the entire transcriptome,3 mass spectrometry then, revolutionary advances in enrichment strategies and (MS)-based proteomics has the advantage of being able to improved performances of capillary liquid chromatography detect and structurally define any covalent changes in a protein (LC) and new MS instrumentation have driven our growing after translation. A daunting number of such changes confer knowledge of many PTMs. In fact, the delineation of the actual altered physiological activity, and many are reversible. There is complexity of many PTMs has emerged mostly through the fi a growing need to carry out accurate measurements of site- past decade. Thus, by signi cant enrichment of classes of fi specific dynamics due to the lack of immunoaffinity reagents for modi ed peptides before MS-analysis, thousands of precise fi fi 5−13 the large numbers of newly identified proteins and their PTM sites can now be identi ed with high con dence. analogs in rewired signaling networks, for example. Thus, the In high-resolution tandem MS, two stages of mass analysis field is seeing an increase in use and further optimization of are used in a single experiment. The MS1 scan refers to the m/z multiplexed targeted, selected-component quantitation by of the precursor ion (peptide or protein), whereas the MS2 spectral acquisition in millisecond time frames. In fact, studies scans refer to the m/z values recorded for their fragmented ionic products. Major developments have led to new of large scale PTM dynamics will be driven by mass spectral- instrumentation that provides both high resolution MS and based quantitationthe methodology of choice. PTMs high mass measurement accuracies for both MS1 and MS2 increase the functional diversity of proteins by adding covalent fi levels simultaneously. The selection of appropriate energy modi cations such as phosphorylation, ubiquitination, glyco- deposition methods, however, is necessary to ensure generation sylation, methylation, and acetylation. Beside single PTMs, fi of sequence ion series required for unambiguous site proteins are often modi ed through a combination of post- assignments. Having high mass spectral measurement quality translational hydrolytic cleavages and the addition of functional has increased the reliability and efficiency of PTM identification groups through a stepwise processes leading to protein at the peptide level and, in addition, has permitted the precise maturation or activation. Protein modifications influence and fi many times even de ne a large variety of normal and fi pathogenic cell biology functions. Therefore, identifying and Special Issue: Post-Translational Modi cations understanding PTMs is critical for gaining a comprehensive Received: November 5, 2014 understanding of cell biology, the detection and delineation of Accepted: December 26, 2014 molecular defects underlying human and other diseases, drug Published: December 26, 2014 © 2014 American Chemical Society 63 DOI: 10.1021/cb500904b ACS Chem. Biol. 2015, 10, 63−71 ACS Chemical Biology Reviews localization of modified sites for some intact protein endoproteinase Glu-C may be employed to increase the PTM sequences.14 In particular, MS fragmentation strategies that coverage.22 generate sufficient peptide fragmentation information are Ionic Interaction-Based PTM Enrichment Strategies. essential for precise PTM identification and localization, by Among all PTMs phosphorylation is the most extensively definition. Among the different fragmentation strategies studied and approximately 197 000 human phosphorylation commonly employed, electron capture and transfer-based sites have been reported.23 Global analysis of serine- and fragmentation techniques have proven to be essential for the threonine-phosphorylation is commonly achieved by metal ion- localization of labile modifications as well as in dramatically based enrichment methods such as immobilized metal affinity 24,25 expanding our experimental capability to carry out sequence chromatography (IMAC) and titanium dioxide (TiO2). analyses on large peptides or even medium size intact proteins. Both approaches use metal cations to bind the negatively Labile structures include many serine or threonine phosphor- charged phosphopeptides, and protocols designed to achieve ylation sites as well as very labile modifications such as O-linked higher enrichment efficiencies have been constantly improved N-acetylglucosamine (O-GlcNAc), γ-carboxyglutamic acid, and − over the past decade. Space does not permit discussion of the others.15 18 extensive literature here, but the interested reader is referred to In this review, we first describe how enrichment strategies the recent Engholm-Keller and Larsen review.27 and the revolutionary advances in mass spectrometry have Multiply phosphorylated peptides, however, remain challeng- contributed to high confidence PTM identification as well as ing to detect and assign unambiguously. The combination of fi site-speci c localization. We then emphasize the discovery of both IMAC and TiO2 enrichment methods, which is termed previously unknown biological roles of PTMs including the sequential elution from IMAC (SIMAC) enables efficient signaling pathway activation role of arginine methylation and separations of monophosphorylated from multiply phosphory- multiple cross-talk forms between PTMs. Finally, we address lated peptides and thus higher numbers of monophosphor- the biological challenges of defining individual site-specific ylation site identifications.28 The use of graphite powder and functions. The complex fields of extracellular glycosylation and titanium(IV) has been reported to increase the identification of protein lipidation are not covered in this review, but the multiply phosphorylated peptides.29,30 An alternative approach interested reader is referred to recently published reviews on that holds promise to achieve higher phosphopeptide enrich- these topics.19,20 ment levels involve gallium complexes that stabilize the weak PTM Enrichment Strategies. Since regulatory PTMs interaction between phosphoryl moiety and serine or threonine feature substoichiometric site occupancies in many cases, residues.31 specific enrichment techniques are essential to achieve Antibody-Based PTM Enrichment Methods. Global phos- detection and characterization of low relative abundance phorylation analysis revealed that 86%, 12%, and 2% of components in digests of cell lysates (Table 1). Common phosphorylation events occur on serine, threonine, and tyrosine, respectively.32 Although phosphotyrosine residues Table 1. PTM Enrichment Strategies represent only a small percentage of all phosphosites the signal- initiating role of receptor tyrosine kinase (RTK) phosphor- enrichment strategy PTM ylation initiates key signaling cascades and plays a driving role antibody-based tyrosine phosphorylation32,33 36 in multiple diseases including cancer. To achieve higher arginine/lysine methylation identification coverage of phosphotyrosine-containing peptides, lysine acetylation10 12,35 antibody-based enrichments in combination with LC-MS/MS ubiquitin-like analyses have been applied yielding quantitative profiling of 33,34 ionic interaction-based serine/threonine/arginine− phosphorylation24
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