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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

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			Action	DRS
	20220901-YP202222554-C1.raw	Raw
	20220901-YP202222554-C2.raw	Raw
	20220901-YP202222554-C3.raw	Raw
	20220901-YP202222554-C4.raw	Raw
	20220901-YP202222554-C5-3.raw	Raw

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Project description:A whole genome screen was used to assay every gene of Escherichia coli strain BW25113 to identify genes involved in susceptibility to the monobactam (beta-lactam) antibiotic aztreonam. The methodology has been called TraDIS-Xpress, and is a version of TraDIS or Tn-seq. A transposon mutant library consisting of several hundred thousand mutants was constructed using a Tn5-derived transposon incorporating an inducible outward transcribing promoter. All the mutants were grown in LB broth cultures supplemented with aztreonam at 2 x, 1 x, 0.5 x and 0.25 x MIC with induction of the transposon promoter using 0.2 mM IPTG or 1 mM IPTG or without induction. Following growth, mutants with increased susceptibility show reduced numbers and those with reduced susceptibility show increased numbers. Each condition was performed in duplicate. The methodology enable genes to be assayed by insertional inactivation or by changes in expression. Expression changes result from altered transcription from upstream transposon insertions transcribing into the gene, or downstream insertions transcribing into the gene in the reverse direction leading to RNA interference through the generation of reverse and complementary RNA. Thus, essential genes into which transposon insertions are not tolerated may be assayed also by changes in numbers of upstream or downstream insertion mutants. Changes to high throughput sequencing protocols permit the generation of nucleotide sequence reads from the known transposon sequences into the surrounding insertion site for all the mutants in the mixture simultaneously. Matching the sequence of the reads to the genome nucleotide sequence of E. coli BW25113 then allows the precise locations of all the transposon insertion sites of all the mutants to be mapped simultaneously. The relative changes in mutants between control (without) and selective condition (with aztreonam) then indicates which genes are involved in susceptibility. The numbers of sequence reads that match is reflected by the number of mutants, and so the degree of susceptibility can also be estimated.

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Staphylococcus aureus linezolid processing proteomic data

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