IDENTIFYING PROTEINS FROM 2-DIMENSIONAL GELS BY MOLECULAR MASS SEARCHING OF PEPTIDE-FRAGMENTS IN PROTEIN-SEQUENCE DATABASES

A rapid method for the identification of known proteins separated by t wo-dimensional gel electrophoresis is described in which molecular mas ses of peptide fragments are used to search a protein sequence databas e. The peptides are generated by in situ reduction, alkylation, and tr yptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted lase r desorption/ionization mass spectrometry of the unfractionated digest . A computer program has been developed that searches the protein sequ ence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates a re included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneou s processing of a large number of two-dimensional gel spots. The metho d was applied to a two-dimensional gel of a crude Escherichia coli ext ract that was electroblotted onto poly(vinylidene difluoride) membrane . Ten randomly chosen spots were analyzed. With as few as three peptid e masses, each protein was uniquely identified from over 91,000 protei n sequences. All identifications were verified by concurrent N-termina l sequencing of identical spots from a second blot. One of the spots c ontained an N-terminally blocked protein that required enzymatic cleav age, peptide separation, and Edman degradation for confirmation of its identity.