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Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.

ABSTRACT: Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissues. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of <5%. Compared to no fractionation prior to LC-MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four-fold. We found that approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.

SUBMITTER: Warren CM

PROVIDER: S-EPMC2885575 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.

Warren Chad M CM Geenen David L DL Helseth Donald L DL Xu Hua H Solaro R John RJ

Journal of proteomics 20100412 8

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochond ...[more]

PMID: 20394843

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Dataset Information

Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.

Publications

Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.

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