MassIVE MSV000090172

Description

R5 was cultured in 10 mL Luria broth with 1.0 mM resveratrol or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform until the cultures reached an average O.D. of 1.0 at 600 nm. Five replicate cultures were used for metabolomics experimentation. The samples were centrifuged at 5,000 x g for 10 min., washed once in 1 mL phosphate buffered saline, centrifuged at 5,000 x g for 10 min., resuspended in 1 mL of 1:1:1:1 acetone/acetonitrile/methanol/water and 1,2500 pmol prednisone, and pulverized in the bead mill. Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without resveratrol). The milled extracts were centrifuged for 20 minutes at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 ul 50% methanol/0.1% formic acid. Fifteen ul of each sample was pooled to create a QC sample. Five ul of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Four subsequent injections of the QC were performed to generate MS2 spectra. After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank1, QC1, sample replicates 1, Blank2, QC2, sample replicates 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. [doi:10.25345/C57659K4X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: resveratrol

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