A STRATEGY FOR THE IDENTIFICATION OF PROTEINS LOCALIZED TO SUBCELLULAR SPACES - APPLICATION TO ESCHERICHIA-COLI PERIPLASMIC PROTEINS

A strategy for the identification of proteins localized to subcellular spaces is illustrated. Proteins in the periplasmic space of E. coli w ere enriched by the procedure of Neu and Hoppel, and the complicated p rotein mixture was partially fractionated by using strong anion exchan ge chromatography [1]. An aliquot from each fraction was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) w ith high sensitivity silver staining for protein detection. An additio nal aliquot of the mixture was digested with the enzyme trypsin and th en analyzed by using microcolumn liquid chromatography electrospray io nization tandem mass spectrometry (LC/MS/MS), employing an instrument control program to automatically acquire tandem mass spectra. The tand em mass spectra were then used to search the E. coli sequence database . Approximately 159 bands appeared on the gels for the enriched protei ns. A total of 80 proteins were identified by LC/MS/MS from the chroma tographic fractions. (C) 1997 Elsevier Science B.V.