Protein Extraction, Digestion, and Labeling With TMT Reagents

SC19 and ΔmnmE cells at mid-log phase were cultured in THB as described above. Three independent biological replicates of bacterial pellets were then treated with SDT buffer (4% SDS,100 mM Tris-HCl, 1 mM DTT, pH 7.6) and heated for 15 min at 100°C. The cell suspensions were sonicated for 5 min (10 s of sonication with 15 s intervals) on ice and the protein concentration in the supernatants was determined using the Bradford protein assay. Each sample (200 μg) were digested with 3 μg of trypsin (Sigma-Aldrich Corporation) at 37°C for 16 h. The resulting tryptic peptides were labeled according to the protocol of TMT Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). The labeled peptides were combined and fractionated by strong cation exchange (SCX) chromatography.