.20 S Proteasome Rapid Separation And Heterogeneity And Protein In Vitro Methylation And Behavioral Characterization

The 20S core particle (CP) is composed of 28 subunits arranged in four stacked heptameric rings (a7(37(37a7) forming a symmetrical barrel-shaped structure. Typically, in previous reports, the purification of CP mainly relied on the antibody, liquid chromatography and affinity tag-based strategies. In this study, we report a relatively simple, economical and effective protocol for proteomic analyses of CP, which only combines differential centrifugations with native-PAGE. Furthermore, it is compatible with both scaled-up purification from erythrocytes and scaled-down purification from low to around 3.0×10'pancreatic cancer cells, SW1990. This protocol was also applied for CP isolation from different species, such as yeast and mouse liver. In addition, a direct three-dimensional gel electrophoresis (d3-DE) approach that omits the interval procedure between native-PAGE and IEF/SDS-PAGE was developed. The results obtained in this study show that this protocol has a valuable potential for the studies of CP and other protein complexes. It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were seventeen proteolytic peptides with twenty modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior and characteristics of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, thus complementary confirmation for in vivo methylation, as well as the precautious guide for the use of methanol in proteomic and in vivo methylation studies.