S. aureus isolate was inoculated and cultured to exponential growth phase (OD600 at 0.5) in TSB and divided into two groups, the control group and the drug treated group. D-3263 was added to the drug treated group until a final concentration of 1/2× MIC and 1/4× MIC reached, and DMSO added in the control group. Then, cultures in both groups were cultivated for 2 h at 200 rpm. The microbial was centrifuged at 12000 rpm for 10 min, washed twice with Phosphate Buffer Saline (PBS). Lyse the cells by bead-beating at 4 °C in RIPA lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS), The protein reduction, alkylation and tryptic digestion were followed as filter-aided sample preparation (FASP). The digestion products were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive plus mass spectrometer.