Dataset Information

Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy.

ABSTRACT: The development of effective strategies for the comprehensive identification and quantification of proteoforms in complex systems is a critical challenge in proteomics. Proteoforms, the specific molecular forms in which proteins are present in biological systems, are the key effectors of biological function. Thus, knowledge of proteoform identities and abundances is essential to unraveling the mechanisms that underlie protein function. We recently reported a strategy that integrates conventional top-down mass spectrometry with intact-mass determinations for enhanced proteoform identifications and the elucidation of proteoform families and applied it to the analysis of yeast cell lysate. In the present work, we extend this strategy to enable quantification of proteoforms, and we examine changes in the abundance of murine mitochondrial proteoforms upon differentiation of mouse myoblasts to myotubes. The integrated top-down and intact-mass strategy provided an increase of ?37% in the number of identified proteoforms compared to top-down alone, which is in agreement with our previous work in yeast; 1779 unique proteoforms were identified using the integrated strategy compared to 1301 using top-down analysis alone. Quantitative comparison of proteoform differences between the myoblast and myotube cell types showed 129 observed proteoforms exhibiting statistically significant abundance changes (fold change >2 and false discovery rate <5%).

SUBMITTER: Schaffer LV

PROVIDER: S-EPMC6201694 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy.

Schaffer Leah V LV Rensvold Jarred W JW Shortreed Michael R MR Cesnik Anthony J AJ Jochem Adam A Scalf Mark M Frey Brian L BL Pagliarini David J DJ Smith Lloyd M LM

Journal of proteome research 20180918 10

The development of effective strategies for the comprehensive identification and quantification of proteoforms in complex systems is a critical challenge in proteomics. Proteoforms, the specific molecular forms in which proteins are present in biological systems, are the key effectors of biological function. Thus, knowledge of proteoform identities and abundances is essential to unraveling the mechanisms that underlie protein function. We recently reported a strategy that integrates conventional ...[more]

PMID: 30180576

Similar Datasets

Project description:Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational protein modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying all protein sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging.MethodsIntact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified.ResultsFrom a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation.ConclusionBy combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.

Dataset Information

Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy.

Publications

Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets