DETECTION OF POLYPEPTIDE ALTERATIONS DURING HEPATOCARCINOGENESIS

主二维聚丙烯酰胺凝胶电泳（2D-PAGE）

The master 2-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) database of RLE cellular polypeptides has been expanded to include detailed information concerning 1100 nucleoplasmic (cytosolic) and 850 particulate associated [35S]-methionine labeled as well as 215 nucleoplasmic and 269 particulate associated [32P]-orthophosphate labeled nuclear polypeptides, respectively. The N-terminal amino acid microsequencing of polypeptides directly from 2D-PAGE gels corresponding to 5 pmol of protein routinely yielded reliable sequence information of 10-15 amino acid residues. Comparison of silver stained, whole cell lysate and purified nuclear polypeptides from adult whole liver tissue indicated a high degree of polypeptide pattern similarity (80-90%) in whole cell lysate and purified nuclear preparations from adult whole liver tissue and from the non-tumorigenic Chang and WRL-68 cell lines. Marked polypeptide differences were observed between normal liver and the tumorigenic HepG2 and Huh-7 cell lines. The 2D-PAGE patterns of the HepG2 and Huh-7 cells were almost completely super-impossible. 2D-PAGE results suggest that the Chang and WRL-68 cells provide valuable in vitro systems for subsequent detailed biochemical studies including cell cycle regulation, growth factor modulation, signal transduction, and apoptosis, in normal versus neoplastic liver tissues. On-line computer linkage of the Elsie 5 analysis system with the SWISS 2D-PAGE human liver protein map revealed >80% matching of proteins, including 50 previously characterized polypeptides. In order to complement the SWISS 2D-PAGE human liver reference map, N-terminal microsequence information of 72 HepG2 nuclear cytosolic polypeptides has been obtained. Nineteen corresponded to known proteins, 35 yielded N-terminal amino acid sequencing data but were not found in the PIR or SWISSPROT databases, while 18 were N-terminally blocked. These results suggest that with the high degree of microsequencing sensitivity and efficiency currently available, it is possible to identify a large percentage of polypeptides directly from 2D-gels.