Analysis of Global Responses by Protein and Peptide Fingerprinting of Proteins Isolated by Two‐Dimensional Gel Electrophoresis

A set of 8 proteins (SSI, sulfate-starvation-induced proteins) was observed by comparative two-dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source. These proteins were isolated after two-dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI2, 5, 7, 8) to be identified with a high degree of confidence. To identify the other SSI proteins, and to obtain sequence information for as many of the proteins as possible, automated on-line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5' region of the Escherichia coli genome. 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 responses. MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material. N-terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.