• Bacillus subtilis;
  • Gel-free analysis;
  • Mass spectrometry;
  • Membrane subproteome;
  • Membrane-embedded peptides


The analysis of integral membrane proteins (IMPs) with mass spectrometry-centered technologies has undergone great progress during the past few years, allowing for the analysis of several hundreds of IMPs. In this study, we investigated three promising shotgun approaches for the identification of IMPs of the model organism Bacillus subtilis. One comprises a classical membrane preparation procedure with carbonate and high-ionic-strength buffers, followed by SDS-PAGE and LC-MS/MS analysis. The two others are based on enzymatic trimming of the crude membrane fraction either with trypsin or proteinase K and subsequent gel-free analysis. As a result, we observed the highest degree of complementarity between the gel-based and the proteinase K approach, since the first exclusively addresses soluble loops and domains of IMPs and gave rise to 8709 unique peptides, whereas the latter contributed 1180 unique peptide identifications from otherwise inaccessible transmembrane helices (TMHs). All three methods contribute significant numbers (381, 284, and 276, respectively) to the total of 527 IMP identifications from the membrane fraction of exponentially growing B. subtilis cells, thus representing approximately 69% of all transcribed IMPs.