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Mass spectrometry-based detection and assignment of protein posttranslational modifications.

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.

SUBMITTER: Doll S

PROVIDER: S-EPMC4301092 | biostudies-other | 2015 Jan

REPOSITORIES: biostudies-other

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Project description:Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes identification and quantification of proteins and the characterization of protein modifications, such as posttranslational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides before MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications, mainly because of the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a "bird's-eye" view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the nonergodic dissociation methods, such as electron-capture dissociation (ECD), greatly enhances the top-down capabilities. ECD is especially useful for mapping labile posttranslational modifications that are well preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research, with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full-sequence coverage, discovering unexpected modifications, identifying and quantifying positional isomers, and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of the top-down method, with a focus on its application in cardiovascular research.

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Mass spectrometry-based detection and assignment of protein posttranslational modifications.

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