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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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  • 1Department 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.

Copyright 2010 Elsevier B.V. All rights reserved.

PMID:
20394843
[PubMed - indexed for MEDLINE]
PMCID:
PMC2885575
Free PMC Article
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