Journal
JOURNAL OF PROTEOMICS
Volume 73, Issue 8, Pages 1551-1561Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jprot.2010.03.016
Keywords
Ischemia; Myocardium; Differential centrifugation; Sarcomere; MassMatrix
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Funding
- NHLBI NIH HHS [R01 HL22231, P01 HL 62426, P01 HL062426, P01 HL062426-10, R01 HL022231, R01 HL022231-32, P01 HL062426-100001] Funding Source: Medline
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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 1 h 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. (C) 2010 Elsevier B.V. All rights reserved.
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