DOI: 10.25345/c5ns0m919
title: MassIVE MSV000096150 - Pseudomonas savastanoi pv. phaseolicola race 5 treated with genistein, daidzein, and DMSO
title: P. savastanoi pv. phaseolicola isolates race 5 (R5) was cultured in 10 mL Luria broth with 400 uM genistein, 400 uM daidzein, or without but with 50 uL DMSO at 28 C at 200 rpm on a shaking platform until the control cultures reached late log-phase growth measured at an optical density of 0.75-0.90 at 600 nm wavelength of visible light. Five replicate cultures of each treatment 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 1,250 pmol prednisone, and pulverized with 0.5 mm glass beads with a Qiagen PowerLyzer 24 bead mill (Qiagen, Hilden, Germany) 10 times at 5,000 rpm for 20 sec (cooled on ice for 2 min between cycles). Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without genistein or daidzein). The milled extracts were centrifuged for 20 min 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 quality control (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-min linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per min. 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. Five subsequent injections of the QC were performed to generate tandem mass spectra (MS2). 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 blank 1, QC 1, sample replicate set 1, Blank 2, QC 2, sample replicate set 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%. The samples also were analyzed in negative ion mode at -2,500 V but with the same other settings.