Search

Subcellular fractionation of a complex proteome (cardiac tissue) allows for enrichment of a subset of low abundant proteins. This permits more in depth analysis of the proteome by reducing the complexity. Adult rat cardiac tissue was made ischemic by ligating the left anterior descending coronary artery in vivo for 1 hour with no reperfusion, and the healthy remote area was differentiated from the ischemic tissue by staining with Evans Blue dye after harvesting the heart. A series of differential centrifugation steps produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric fractions of rat ischemic and remote healthy tissue. The sarcomeric fractions of the remote vs ischemic cardiac tissue were digested with trypsin, and the peptides were labeled with isobaric tags for relative quantitation (iTRAQ). The labeled peptides were then fractionated with an Agilent 3100 OFFGEL fractionator, which separated the peptides in 12 fractions based on their isoelectric point from pH 3-10. The 12 fractions from the OFFGEL were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode to detect the low mass reporter ions of the isobaric tags on the peptides. The peptides were analyzed with MASCOT (MatrixScience), and Scaffold v2.5.2 Q+ with a minimum of two unique peptides and a level of confidence set at 95%. Five-fold more proteins were identified when the labeled digests were fractionated with OFFGEL compared to no fractionation prior to LC-MS/MS. With a one hour ischemic event, we found approximately 11% of the detected proteins in the sarcomeric fraction had changed at least 1.5 fold. Therefore, this in-solution method incorporating sub-proteomic fractionation in conjunction with OFFGEL separation may be an approach for discovery of relative protein changes in cardiac tissue.