Microsequencing of proteins recorded in human two-dimensional gel protein databases

Abstract

Sixty-six human proteins recorded in the master transformed human epithelial amnion cells (AMA) (55) and keratinocyte (11) two-dimensional gel protein databases have been microsequenced since the last publication of the AMA database (Electrophoresis 1990, 12, 989-1071). Coomassie Brilliant Blue stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes and in situ digested with trypsin. The eluting peptides were separated by reversed-phase high performance liquid chromatography (HPLC), collected individually and sequenced. Computer searches using the FASTA and TFASTA programs from the Genetics Computer Group indicated that 29 of the analyzed polypeptides correspond to hitherto unknown proteins.