Staphylococcus aureus linezolid processing proteomic data

ABSTRACT: Exponentially growing S.aureus ATCC 25923 was treated with linezolid (LZD) at concentrations of 1/2× MIC, 1× MIC, and 5× MIC, or an equivalent amount of DMSO for 1 hour. The bacteria were pelleted by centrifugation and washed twice with saline. The samples were then re-suspended in bacteria lysis buffer (100 mM NH4HCO3, 8 M urea, 1 mM PMSF, 1× Protease Inhibitor Cocktail) and mechanically lysed using 0.1 mm silica beads in a Precellys tissue homogenizer at 4°C. Subsequently, the samples were crushed with a non-contact ultrasonic cell crusher for 1 hour. Silica beads and bacterial debris were removed by centrifugation at 1200×g and 4°C for 20 minutes. The protein concentration in the supernatant was measured using a Bradford assay kit (Beyotime, China). Next, the samples were reduced with 10 mM DTT for 1 hour, followed by alkylation in the dark with 45 mM iodoacetamide to break disulfide bonds. Each sample underwent trypsin digestion, with 50 μg of total protein digested using 2 μg trypsin overnight at 37°C or 1.5 hours at 56°C, and an additional 1 μg trypsin added twice during digestion. The digested peptides were filtered using Ultra Centrifugal Filters (Millipore, USA) and desalted using MonoSpin C18 (Shimadzu, Japan) following the manufacturer's protocol. The peptide samples were dried using a freeze-dryer and resuspended in 25 μl of water with 0.1% formic acid (vol/vol).Data acquisition was performed using a reverse-phase LC-MS/MS system comprising an Easy nLC1200 system (ThermoFisher, USA) coupled with a Q Exactive Plus Orbitrap MS System (ThermoFisher, USA).

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Staphylococcus Aureus

TISSUE(S): Cell Culture

SUBMITTER: Dan Luo  

LAB HEAD: Dan luo

PROVIDER: PXD046938 | Pride | 2023-12-11

REPOSITORIES: Pride