Rapid label‐free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome

Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane‐embedded sub‐proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re‐annotation of the theoretical E. coli IMP regarding the sub‐cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC‐MS/MS analysis, we experimentally identified ～45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label‐free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over‐synthesizing the membrane‐embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.