Abstract:
The heart proteome has been extensively characterized because of its potential to elucidate the molecular mechanisms underlying normal cardiac development and disease pathology. Human cardiac tissue, however, poses unique challenges for proteomic analysis because of its abundance of cardiac myofibrils, which contain some of the largest proteins in the human proteome and are difficult to solubilize. I will present our methodological developments for the improved analysis and quantification of this challenging sample type. Specifically, we investigated the use of detergents, extended digestion, and phase extraction to recover proteins and peptides from the typically discarded pellets of heart tissue lysates. We also demonstrated the feasibility of using spectral counting, a facile and flexible label-free quantitation method, to reveal biological differences and identify biomarkers, particularly maturation markers of stem cell-derived cardiomyocytes, in the resulting heart proteome. Finally, we developed an in-sample calibration approach (Heavy-Match-Light) that enabled accurate quantification of the copy numbers of human cardiac ion channels in the insoluble fraction of heart lysates using only a single isotopologue.