Investigate the Insoluble Fraction in Heart Tissue Preparation after Deoxycholate for Potential Loss of Proteins in Heart Proteomics

Abstract

The heart proteome has been intensively characterized because of its ability to elucidate the molecular mechanisms involved in normal development and disease pathology. Human heart tissue, however, poses unique challenges for proteomic sample preparation, because of the abundance of cardiac myofibrils, which contain some of the largest proteins in the human proteome and are difficult to solubilize. We investigated the insoluble fraction after the use of a popular sodium deoxycholate (SDC) detergent to aid in the solubilization of proteins in human ventricular homogenates to prevent potential protein loss. To bring potential proteins from the insoluble fractions into solution, we investigated the effect of digestion-aided protein solubilization (DAPS). To facilitate downstream data analysis, we demonstrated the possibility of using spectral counting, a facile and flexible label-free quantitation scheme, to quantify proteins identified in a variety of tissue fractions with drastically different matrix backgrounds. Among the differentially distributed proteins and peptides, we discovered that large sarcomere proteins, represented by titin and myosin heavy chains, were enriched in the insoluble fraction of SDC, and our strategy resolved a more comprehensive sarcomere proteome. In addition, acid precipitation of SDC preferentially resulted in the loss of hydrophobic peptides, which might not be significant for identification, but is crucial for quantitation in proteomics.