Sub-proteomic fractionation, iTRAQ, and OFFGEL-LC-MS/MS approaches to cardiac proteomics.
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.
Warren
Chad M
CM
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Geenen
David L
DL
Helseth
Donald L
DL
Xu
Hua
H
Solaro
R John
RJ
D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D017202:Myocardial Ischemia / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism; D053719:Tandem Mass Spectrometry
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