Lc esi qtof

To identify changes in proteins present on the cell surface of MRSA 4423 due to glucose and glabridin, cultures were grown in presence of 1% glucose and supplemented with glabridin (1/4 MIC). Cells grown only with glucose were used as a biofilm positive control and cells grown without both glucose and glabridin were used as a biofilm negative control. After 48 h of growth under static conditions, 5 ml cells of equal OD were collected from each set grown in triplicates, centrifuged at 4,000g for 10 min and supernatant was discarded. Pellets were washed twice with 1X PBS and cells were resuspended in 1 ml of 1X PBS. To collect surface proteins, all cell suspensions were treated with 1 μg/ml trypsin enzyme for 1 h at 37°C (Ythier et al., 2012 (link)). Trypsin shaved proteins in supernatants fraction were collected by centrifugation at 16,000g for 10 min in 4°C and processed as above for identification by LC-ESI-QTOF (Agilent, United States).

The biofilm ECM proteins were extracted using a method as described by Ythier et al. (2012) (link). Briefly, the culture of MRSA 4423 was grown in the presence of glucose for 48 h in static conditions, and non-adherent cells were removed by decanting and rinsing with 1X PBS. To shave off proteins from biofilm ECM, 1 μg/ml trypsin enzyme was used. After 1 h of enzymatic treatment at 37°C, cells were separated by centrifugation at 16,000g, 4°C for 15 min, and partially digested proteins in supernatant were transferred into a new tube and the peptide mix was processed for identification by Liquid Chromatography/High Resolution Mass Spectrometry (LC-ESI-QTOF) (Agilent, United States).