Proteomic Characterization of Human Erythrocyte 20S Proteasome and Analysis of Species-dependent 20S Proteasome Heterogeneity

A method for the purification of 20S proteasome (20S core particle, CP) was developed by combining differential centrifugations with nondenaturing polyacrylamide gel electrophoresis (native-PAGE), irrespective of species origins of CPs. CP purified from human erythrocytes was subjected to proteomic analysis by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), revealing 33 spots of subunit isoforms with different molecular weights and isoelectric points. Furthermore, other four CPs were purified from yeast, mouse liver, and two pancreatic cancer cell lines SW1990 and PANC-1 using the method mentioned above and subjected to proteasome heterogeneity analysis by two-dimensional native/SDS-PAGE (native/sodium dodecyl sulphate polyacrylamide gel electrophoresis), in combination with CP from erythrocytes. In conclusion, the method described acts as a rapid and effective tool for CP isolations, and the results obtained may serve as a footstone for the investigations of species-dependent proteasome heterogeneity.