Figure 2

Workflow for the analysis of membrane proteins via 1D gel LC-MS/MS. The current workflow for analysis of the B. subtilis membrane proteome involves several steps. First bacteria are cultivated under appropriate conditions and samples are withdrawn. Next, the harvested cells are disrupted, and the membrane fraction is enriched in successive centrifugation steps. Membrane proteins are separated by one-dimensional SDS PAGE, excised from the gel, and digested with an appropriate protease. The peptides thus obtained are analyzed by liquid chromatography and mass spectrometry for protein identification. Different approaches can be employed for quantitative membrane proteomics. For B. subtilis, these have so far involved stable istotope labeling with amino acids (SILAC) such as ¹³C6-¹⁵N2 lysine, and ¹⁴N/¹⁵N metabolic labeling.