Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.
The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.
Ward
L D
LD
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Parkville, Australia.
Hong
J
J
Whitehead
R H
RH
Simpson
R J
RJ
D000595:Amino Acid Sequence; D003110:Colonic Neoplasms / Q000737:chemistry*; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D010669:Phenylthiohydantoin; D011145:Polyvinyls; D012394:Rosaniline Dyes; D012697:Serine Endopeptidases; D014407:Tumor Cells, Cultured; D005465:Fluorocarbon Polymers
D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D010669:Phenylthiohydantoin; D011145:Polyvinyls; D012394:Rosaniline Dyes; D012697:Serine Endopeptidases; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C028344:glutamyl endopeptidase; C004692:coomassie Brilliant Blue