Dataset Information

In-depth proteomic analysis of human tropomyosin by top-down mass spectrometry.

ABSTRACT: Tropomyosins (Tms) are a family of highly conserved actin-binding proteins that play critical roles in a variety of processes, most notably, in the regulation of muscle contraction and relaxation. It is well known that different Tm isoforms have distinct functions and that altered expression of Tm isoforms could lead to changes in cardiac structure and function. To precisely define Tm isoform expression in the human heart, towards a better understanding of their functional roles, we have employed top-down mass spectrometry for in-depth proteomic characterization of Tm isoforms. Using a minimal amount of human heart tissue from rejected donor organs, we confirmed the presence of multiple Tm isoforms including ?-Tm, ?-Tm and ?-Tm in the human heart, with ?-Tm being the predominant isoform, followed by minor isoforms of ?-Tm and ?-Tm. Interestingly, our data revealed regional variations of Tm isoforms and post-translational modifications in the human heart. Specifically, the expression level of ?-Tm was highest in the left atrium but nearly undetectable in the left ventricle. The phosphorylation level of ?-Tm (p?-Tm) was significantly higher in the atria than it was in the ventricles. The sequences of all Tm isoforms were characterized and the sites of post-translational modifications were localized. Clearly, top-down mass spectrometry is an attractive method for comprehensive characterization of Tm isoforms and post-translational modifications since it can universally detect and quantify all types of protein modifications without a priori knowledge and without the need for specific antibodies.

SUBMITTER: Peng Y

PROVIDER: S-EPMC3849107 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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In-depth proteomic analysis of human tropomyosin by top-down mass spectrometry.

Peng Ying Y Yu Deyang D Gregorich Zachery Z Chen Xin X Beyer Andreas M AM Gutterman David D DD Ge Ying Y

Journal of muscle research and cell motility 20130724 3-4

Tropomyosins (Tms) are a family of highly conserved actin-binding proteins that play critical roles in a variety of processes, most notably, in the regulation of muscle contraction and relaxation. It is well known that different Tm isoforms have distinct functions and that altered expression of Tm isoforms could lead to changes in cardiac structure and function. To precisely define Tm isoform expression in the human heart, towards a better understanding of their functional roles, we have employe ...[more]

PMID: 23881156

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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

In-depth proteomic analysis of human tropomyosin by top-down mass spectrometry.

Publications

In-depth proteomic analysis of human tropomyosin by top-down mass spectrometry.

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