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.