MassIVE MSV000090171

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 without resveratrol reached an average optical density (O.D.) of 0.78 (mid-log phase growth) at 600 nm. Protein was prepared by centrifuging the cultures at 5,000 x g, washing the cells two times in 5 mL phosphate buffered saline, resuspending the cells in 1 mL 100 mM Tris pH 8.5\1 mM EDTA\10 mM DTT\0.5% n-dodecyl beta-D-maltoside\Proteinase Inhibitor cOmplete [1 tablet/10 mL, Roche Diagnostics, Indianapolis, IN], and pulverizing the cells with 0.5 mm glass beads with a Qiagen PowerLyzer 24 bead mill (Qiagen, Hilden, Germany) 10 times at 5,000 rpm for 20 seconds (cooled on ice for 2 minutes between cycles). The extracts were centrifuged at 10,000 x g for 5 minutes, and the supernatants sent to the Thermo Fisher Scientific Center for Multiplexed Proteomics at the Harvard Medical School. Protein (~300 ug) was precipitated from the supernatants with methanol/chloroform, reduced in Tris(2-carboxyethyl)phosphine, carboxyamidomethylated with iodoacetamide, and digested sequentially with LysC and trypsin using methods previously described (Isasa et al., 2015). Peptides (~120 ug) were labeled with Tandem Mass Tags (TMTpro; Thermo Fisher Scientific) per the manufacturer instructions. Tags 126, 127N, and 127C were assigned to control replicates, and 128N, 128C and 129N were assigned to resveratrol-treated replicates. Samples were analyzed to determine tag label incorporation percentage (>95%) and to estimate quantitative ratios between samples. On the basis of the quantitative ratios, labeled samples were mixed together and fractionated by high-pH reverse phase HPLC (Isasa et al., 2015). Twelve concatenated fractions were analyzed. Peptides from each fraction were separated using a gradient of 3 to 22% of 90% acetonitrile\0.1% formic acid over 150 minutes and were electrosprayed at 2.6 kV into an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) operating in data-dependent mode with positive polarity. Quadrupole isolation was enabled, and survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 350-1500 m/z. The automatic gain control (AGC) target was set to 1,000,000 and the maximum injection time was set to 50 milliseconds (msec). The most abundant precursor ions (minimum intensity threshold 5,000) were fragmented by collision-induced dissociation (35% energy) and fragment ions were detected in the linear ion trap (AGC 15,000, 50 msec maximum injection). Analyzed precursors were dynamically excluded for 180 seconds. Multiple MS2 fragment ions were captured using isolation waveforms with multiple frequency notches and fragmented by high energy collision-induced dissociation (50% normalized collision energy). MS3 spectra were acquired in the Orbitrap (AGC 150,000; maximum injection time 200 msec, 50,000 resolution scanning from 100-1200 m/z). [doi:10.25345/C5BZ61C8R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: resveratrol

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