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Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

ABSTRACT: One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

SUBMITTER: Donnelly DP

PROVIDER: S-EPMC6719561 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

Donnelly Daniel P DP Rawlins Catherine M CM DeHart Caroline J CJ Fornelli Luca L Schachner Luis F LF Lin Ziqing Z Lippens Jennifer L JL Aluri Krishna C KC Sarin Richa R Chen Bifan B Lantz Carter C Jung Wonhyeuk W Johnson Kendall R KR Koller Antonius A Wolff Jeremy J JJ Campuzano Iain D G IDG Auclair Jared R JR Ivanov Alexander R AR Whitelegge Julian P JP Paša-Tolić Ljiljana L Chamot-Rooke Julia J Danis Paul O PO Smith Lloyd M LM Tsybin Yury O YO Loo Joseph A JA Ge Ying Y Kelleher Neil L NL Agar Jeffrey N JN

Nature methods 20190627 7

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using ...[more]

PMID: 31249407

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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

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

Publications

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

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