| The goal of many proteomic investigations is to compare and evaluate the complement of expressed proteins in a cellular structure or physiological state to facilitate discovery-based research and knowledge generation. Limitations in protein separation and identification techniques prevent the simultaneous characterization of an entire proteome, particularly when large differences in protein concentration exist (high dynamic range). These challenges can be addressed by prefractionation of a sample to selectively enrich a set of proteins creating a simplified subproteome. However, prior to employing any subfractionation method, the reproducibly and selectivity must be determined for each cell type. In cardiac tissue, nuclear proteins are difficult to examine because proteins of the contractile apparatus and energy production dominant the heart proteome. This study examined two nuclear protein enrichment strategies specifically for the cardiac muscle nucleus and included: (1) optimized centrifugation based protocol where nuclei were isolated based on their size and buoyant density and (2) a differential detergent fractionation protocol where nuclear proteins are extracted based on physicochemical properties. These approaches exploit different physical properties of nuclear organelle and it proteins and so, we propose they will be complementary in their coverage of the nuclear proteome. To measure the nuclear enrichment and overall composition of the subproteomes, light microscopy, western blot, 2-dimensional electrophoresis and mass spectrometry were performed on frozen rat cardiac tissue and also in liver tissue. In cardiac tissue, although enrichment of the DNA binding protein histone H3 was observed for each protocol, few validated nuclear proteins were identified in the "nuclear" fraction obtained from the centrifugation (16%) and detergent extraction (10%) strategies. This limited enrichment of nuclear proteins may not be suitable for proteomic analysis but rather as a starting point for subsequent targeted affinity based enrichment methods. To achieve purity of the nuclear proteome another method will be required. |
