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Determination of oxidative protein modifications using mass spectrometry.

ABSTRACT: Numerous oxidative modifications to proteins and amino acids have been identified with most susceptible, to varying degrees, of some form of oxidative modification. The consequence of oxidation on protein structure and function reveals that some of these modifications are functionally important. The discovery and accurate characterization/description of existing and new modifications requires modern instrumentation, great care, and attention to detail, especially if the modifications are present in low stoichiometric quantities or they only exist transiently. The focus of this brief review is on the use of mass spectrometry, protein chemistry, and proteomics methods and tools to identify oxidatively modified proteins and peptides along with the characterization of specific sites. Many of the specialized mass spectrometry technologies and techniques are becoming more widely available in research laboratories with mass spectrometry or proteomics facilities allowing even non-expert researchers in the field to accurately determine modifications. Illustrative examples of some approaches are provided from the author's work, collaborative research projects, and elsewhere.

SUBMITTER: Raftery MJ

PROVIDER: S-EPMC6837444 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Determination of oxidative protein modifications using mass spectrometry.

Raftery Mark J MJ

Redox report : communications in free radical research 20140404 4

Numerous oxidative modifications to proteins and amino acids have been identified with most susceptible, to varying degrees, of some form of oxidative modification. The consequence of oxidation on protein structure and function reveals that some of these modifications are functionally important. The discovery and accurate characterization/description of existing and new modifications requires modern instrumentation, great care, and attention to detail, especially if the modifications are present ...[more]

PMID: 24702029

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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.

Dataset Information

Determination of oxidative protein modifications using mass spectrometry.

Publications

Determination of oxidative protein modifications using mass spectrometry.

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