MICROPREPARATIVE IMMOBILIZED PH GRADIENT 2-DIMENSIONAL ELECTROPHORESIS IN COMBINATION WITH PROTEIN MICROSEQUENCING FOR THE ANALYSIS OF HUMAN LIVER PROTEINS

Simplified methodology has been developed for the direct N-terminal am ino acid microsequencing of human liver and hepatoma derived polypepti des, following micropreparative two-dimensional polyacrylamide gel ele ctrophoresis (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) m embranes nearly 100 well resolved Ponceau S stained polypeptides were readily visualized, from which, 32 adult liver S-9 and 72 HepG2 nuclea r cytosolic polypeptides were subjected to N-terminal microsequencing. Twenty normal adult liver and 54 HepG2 polypeptides yielded N-termina l sequence information, of which 17 and 19 polypeptides, respectively, exhibited high sequence homology to previously identified proteins. T he initial yields of the proteins sequenced ranged from 2-14 pmols and yielded sequences of 14-26 amino acid residues. Many of the adult liv er and HepG2 proteins contained inferred leader sequences since the fi rst sequenced residue was several (20-30) residues from the methionine initiation site predicted by the cDNA of the adult liver. Quantitativ e comparison of 60 well characterized hepatic proteins between normal adult liver and two nontransformed, Chang and WRL-68, and four human h epatoma derived cell lines, HepG2, Huh-7, FOCUS, and SK-Hep, revealed a high homogeneity of protein expression both qualitatively and quanti tatively in both whole cell lysate and purified nuclear preparations. Most notable differences include the previously characterized polypept ides: carbamoyl phosphate synthase, MER5 homologous protein, cytidylat e kinase, phosphatidylethanolamine-binding protein and mitochondrial e noyl-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 t he nontransformed, Chang and WRL-68, cell lines but not expressed or e xpressed at greatly decreased levels in each of tumor derived liver ce ll lines. Pyruvate carboxylase, superoxide dismutase, serotransferrin, liver fatty acid binding protein, 1-hydroxyprostaglandin dehydrogenas e, NADH dehydrogenase (ubiquinone) as well as three N-terminally block ed polypeptides: 9 (57/6.00), 53 (24/4.90) and 63 (16/4.70) were detec ted only in whole adult liver tissue and not in any of the cultured ce ll lines. Two additional polypeptides: U35, (27/6.05) and 58 (22/5.70) yielded N-terminal partial amino acid sequences but were not identifi ed in established protein databases. We have shown that micropreparati ve IPG 2-D PAGE in combination with protein microsequencing provides a convenient one step procedure to rapidly obtain partial amino acid se quence information for nearly 100 individual polypeptides directly fro m a single 2-D PAGE gel with numerous applications to a wide variety o f biological model systems.