Proteomic Analysis of Heart Tissue

Heart tissue was cut into 1 mm pieces, washed with PBS containing a protease inhibitor cocktail (5 mM EDTA, 100 µM PMSF, 100 µM TLCK, 100 µM TPCK, 1 µM pepstatin A, 100 µM leupeptin) at 4°C, and centrifuged three times at 10,000 rpm with supernatant discarding. Lysis buffer (7 M urea, 2 M thiourea, 2% Triton X-100, 1% DTT and the protease inhibitor cocktail) was then added in the proportion of 6 mL to 1 g of tissue, followed by vortexing for 2 min and sonication in a refrigerated bath for 2 min three times. The material was left at 4°C for 20 min and finally centrifuged at 14,000 rpm for 15 min. The supernatant (heart extract) was submitted to protein quantitation using the PlusOne 2D Quant Kit (GE Life Sciences). The protein profiles of heart extracts (100 µg) were analyzed by 2DE [100] (link). Peptide mass fingerprinting [63] (link) was used for protein identification via an Autoflex II mass spectrometer (Bruker Daltonics) and the Mascot software (Matrix Science).