Quantitation of de novo localized 15N-labeled lipoproteins and membrane proteins having one and two transmembrane segments in a Bacillus subtilis secA temperature-sensitive mutant using 2D-PAGE and MALDI-TOF MS

We developed a means of quantifying proteins that have just localized in the cytoplasmic membrane using N-15-whole cell labeling together with 2D-PAGE and MALDI-TOF MS. The localization of 18 among 20 proteins consisting of 8 lipoproteins, 11 integral membrane proteins having one or two transmembrane segments and one secretory protein in the membrane fractions of Bacillus subtilis, was inhibited by the absence of SecA in a temperature-sensitive mutant. The time course of inhibition indicated that SecA participates in the localization of those proteins through immediately dependent, delayed dependent, and independent ways.