The wildtype and lsr2 insertion mutation strains of M. smegmatis were culturedgrown in 7H9 medium until reachingto an OD600 of 1.0 and then collected. The cells were then collected, re-suspended with SDT lysis buffer (4% SDS, 10 mM DTT, 100 mM TEAB), and ultrasonicated on ice for 5 min. Subsequently, the supernatant was reduced with 10 mM DTT for 1 h at 56 ℃, then alkylated with sufficient iodoacetamide in the dark for 1 h at room temperature. After that, the samples were treated with precooled acetone, centrifuged, and dissolved with a dissolution buffer (8 M Urea, 100 mM TEAB, pH 8.5). The proteins were quantified, digested with Trypsin overnight in the presence of CaCl2, finally tagged with acetonitrile-dissolved TMT labeling reagent. Peptide fractions were separated using a C18 column (Waters BEH C18, 4.6×250 mm, 5 μm) on a Rigol L3000 HPLC system. The separated peptides were analyzed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with a Q ExactiveTM HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode. The resulting spectra from each run were searched separately against a database containing M. smegmatis mc2 155 protein sequences using the search engine Proteome Discoverer 2.4 (Thermo). treated and analyzed according to the previous procedures 51. The protein quantitation results were sStatistically analyzed analyses were evaluated by t-test assays. Proteins that showed significantly differences in quantitation between the experimental and control groups, (with p value < 0.05 and |log2FC| > 0.6) were defined as differentially expressed proteins.