Micropreparative immobilized pH gradient two-dimensional electrophoresis in combination with protein microsequencing...

Simplified methodology has been developed for the direct N-terminal amino acid microsequencing of human liver and hepatoma derived polypeptides, following micropreparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Utilization of immobilized pH gradient (IPG) gel strips in the first dimension permitted protein loading of 0.5-2.0 mg with negligible diminution of polypeptide resolution. Following 2-D separation and electrotransfer to polyvinylidene difluoride (PVDF) membranes nearly 100 well resolved Ponceau S stained polypeptides were readily visualized, from which, 32 adult liver S-9 and 72 HepG2 nuclear cytosolic polypeptides were subjected to N-terminal microsequencing. Twenty normal adult liver and 54 HepG2 polypeptides yielded N-terminal sequence information, of which 17 and 19 polypeptides, respectively, exhibited high sequence homology to previously identified proteins. The initial yields of the proteins sequenced ranged from 2-14 pmols and yielded sequences of 14-26 amino acid residues. Many of the adult liver and HepG2 proteins contained inferred leader sequences since the first sequenced residue was several (20-30) residues from the methionine initiation site predicted by the cDNA of the adult liver. Quantitative comparison of 60 well characterized hepatic proteins between normal adult liver and two nontransformed, Chang and WRL-68, and four human hepatoma derived cell lines, HepG2, Huh-7, FOCUS, and SK-Hep, revealed a high homogeneity of protein expression both qualitatively and quantitatively in both whole cell lysate and purified nuclear preparations. Most notable differences include the previously characterized polypeptides: carbamoyl phosphate synthase, MER5 homologous protein, cytidylate kinase, phosphatidylethanolamine-binding protein and mitochondrial enoyl-CoA hydratase as well as three N-terminally blocked polypeptides: 11 (63 kDa/pI 7.00), 56 (26/6.45) and 59 (22/6.00) all of which were expressed at similar levels in normal adult liver tissue and each of the nontransformed, Chang and WRL-68, cell lines but not expressed or expressed at greatly decreased levels in each of tumor derived liver cell lines. Pyruvate carboxylase, superoxide dismutase, serotransferrin, liver fatty acid binding protein, 1-hydroxyprostaglandin dehydrogenase, NADH dehydrogenase (ubiquinone) as well as three N-terminally blocked polypeptides: 9 (57/6.00), 53 (24/4.90) and 63 (16/4.70) were detected only in whole adult liver tissue and not in any of the cultured cell lines. Two additional polypeptides: U35, (27/6.05) and 58 (22/5.70) yielded N-terminal partial amino acid sequences but were not identified in established protein databases. We have shown that micropreparative IPG 2-D PAGE In combination with protein microsequencing provides a convenient one step procedure to rapidly obtain partial amino acid sequence information for nearly 100 individual polypeptides directly from a single 2-D PAGE gel with numerous applications to a wide variety of biological model systems.