ANALYSIS OF GLOBAL RESPONSES BY PROTEIN AND PEPTIDE FINGERPRINTING OFPROTEINS ISOLATED BY 2-DIMENSIONAL GEL-ELECTROPHORESIS - APPLICATION TO THE SULFATE-STARVATION RESPONSE OF ESCHERICHIA-COLI

A set of 8 proteins (SSI, sulfate-starvation-induced proteins) was obs erved by comparative two-dimensional electrophoresis to be induced whe n Escherichia coli were grown using compounds other than sulfate or cy steine as the sole sulfur source, These proteins were isolated after t wo-dimensional gel electrophoresis, digested with trypsin and the mass es of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to s earch the databases, allowing four of the SSI proteins (SSI2, 5, 7, 8) to be identified with a high degree of confidence. To identify the ot her SSI proteins, and to obtain sequence information for as many of th e proteins as possible, automated on-line HPLC MS/MS (fragmentation an alysis using coupled mass scanning devices) data collection was perfor med. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 a nd 3) were identified in the 8.5' region of the Escherichia coli genom e. N-terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus subtilis orfM gene product. SSI 4 was not found in the databases by any of these methods. The methods described are of general use for the rapid analysis of complex cell r esponses. MS data accumulation takes about 5 min/protein for protein f ingerprinting and 30 min for peptide fingerprinting and requires appro ximately 100 fmol of material. N-terminal sequencing however, takes ab out 5 h/protein and approximately 1 pmol to obtain a 10 amino acid seq uence for a search.