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

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.