(342g) Detecting Low Abundance Proteins in Skinned Cardiac Myofibrils

Skinned cardiac myofibrils are the most commonly utilized muscle system to investigate muscle physiology. However, other than the well established components of the skinned myofibrils very little is known about the low abundance proteins present in skinned myofibrils. In 2005, our group found that a kinase was present in the skinned fiber which phosphorylated troponin I under certain conditions (Gomes et al. J. Mol. Cell. Cardiology 2005, 39:754-65). Western blotting has suggested the presence of the kinase cAMP dependent Protein kinase A (PKA) as well as the proteolytic complex, the proteasome, in the skinned myofibrils. Electrophoresis followed by trypsin digestion of gel pieces and LC-MS/MS is a powerful method for detecting proteins and associating partners (Gomes et al. Mol. Cell Proteomics 2009, 8: 302-315). One dimensional electrophoresis, trypsin digestion of the gel pieces and LC-MS/MS showed the presence of many proteins in skinned fibers but no kinases or proteasomes. The main problem is the large dynamic range of protein concentrations in the skinned fiber. Actin and myosin together make up more than 40% of the skinned fiber and dominate detection by mass spectrometry. To detect the medium to low abundant proteins the Proteominer protein enrichment kit was utilized. The Proteominer technology is based on the treatment of complex protein samples with a large, highly diverse library of hexapeptides bound to chromatographic supports. Each unique hexapeptide should bind to a unique protein sequence resulting in highly abundant proteins saturating their ligands, with excess protein being washed out and low abundance proteins being concentrated, ultimately reducing the dynamic range of the proteins in the sample. Few of the proteins present on cardiac myofibrils are released by homogenization in PBS so detergents (NP40, Triton X-100, SDS) are utilized to extract proteins. One problem with the use of hexapeptides is that they have not been optimized for samples containing detergents or urea. No method is currently available for using the hexapeptide beads on samples exposed to detergent. Isolation of the myofibrillar proteins in SDS containing solution followed by dialysis in PBS results in precipitation of many of the proteins. Several attempts to find a hexapeptide compatible medium that solubilize the myofibrillar proteins resulted in two suitable candidates: hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE). HFIP and TFE are typically utilized to solubilize proteins. While both solvents were found to be useful: TFE and HFIP gave similar results, but TFE was found to be easier to use. After solubilization in SDS buffer, samples are precipitated with TCA/acetone and homogenized in Tris buffered TFE (40% v/v). This procedure resulted in most of the proteins being resolubilized in the Tris buffered TFE which was compatible with the hexapeptide beads. Porcine skinned fibers analyzed by the hexapeptide beads showed distinct changes in the banding pattern on one dimensional gels, suggesting that medium to low abundant proteins were enriched by this method. LC-MS/MS confirmed that medium to low abundant proteins were enriched by the Proteominer hexapeptide beads. It is also important to note that the high concentrations of proteins recommended for incubating the hexapeptide beads in the Proteominer manual (>50mg/ml) are not required for high protein retrieval from the beads.