Quantitative proteomics of sub-cellular compartments of M. smegmatis

We performed a comprehensive proteomic analysis of the subcellular fractions of M. smegmatis. The proteomes of 4 independent cultures of M. smegmatis were split in 4 subcellular fractions: plasma membrane (PM), culture filtrate (secretome, CF), mycomembrane-containing cell-wall (CW) and cytoplasm (CP). PM and CW were isolated from the cell lysates by differential ultracentrifugation on sucrose gradient. After concentration on SDS-PAGE, proteins from each fraction were digested in gel using trypsin. Peptides were then extracted from the gel and analyzed in quadruplicate injections by nano Liquid Chromatography coupled to tandem mass spectrometry (NanoLC-MS/MS) using an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific). MS data were searched against the Uniprot M. smegmatis mc²155 database for protein identification and the quantitative comparison of relative protein abundances was performed using MS1-based label-free quantification. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017602.