A strategy for the identification of proteins localized to subcellular spaces: Application to E. coli periplasmic proteins

Abstract

A strategy for the identification of proteins localized to subcellular spaces is illustrated. Proteins in the periplasmic space of E. coli were enriched by the procedure of Neu and Hoppel, and the complicated protein mixture was partially fractionated by using strong anion exchange chromatography [1]. An aliquot from each fraction was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with high sensivity silver staining for protein detection. An additional aliquot of the mixture was digested with the enzyme trypsin and then analyzed by using microcolumn liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS), employing an instrument control program to automatically acquire tandem mass spectra. The tandem mass spectra were then used to search the E. coli sequence database. Approximately 159 bands appeared on the gels for the enriched proteins. A total of 80 proteins were identified by LC/MS/MS from the chromatographic fractions.