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J Proteomics. 2010 Jun 16;73(8):1551-61. doi: 10.1016/j.jprot.2010.03.016. Epub 2010 Apr 12.

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

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1
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, USA.

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.

PMID:
20394843
PMCID:
PMC2885575
DOI:
10.1016/j.jprot.2010.03.016
[Indexed for MEDLINE]
Free PMC Article
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