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Project description:Monobromobimane (mBBr) labelling: mBBr labelling was carried out either immediately prior to 42°C, two hour heat shock, or immediately following heat shock. Medium was removed from culture flasks and cells were washed using PBS. Cells were then labelled by incubation at 37°C with 6 mL of 400 µM mBBr (Sigma-Aldrich, #B4380) for 10 minutes. Following labeling, 6 ml of 2 mM L-glutathione reduced (GSH, SigmaAldrich, #G4251) in PBS was added to quench the mBBr reaction. The quenched mBBr solution was removed and cells were washed with PBS. Mass spectrometry (MS) sample preparation, analysis and data processing: Six 1.6 mm steel beads (Next Advance Inc.) were added to the cell pellet tube with 30 µL SL-DOC (1.1% (w/w) sodium dodecyl sulfate (Sigma), 0.3% (w/w) sodium deoxycholate (Sigma), 25 mM ammonium bicarbonate (AB, Fluka), in de-ionised (DI) water containing 0.1% (v/v) protease inhibitor cocktail (Sigma), and 0.1% (v/v) phosphotase inhibitor cocktail (Sigma). Cells were homogen
2021-06-15 | PXD025329 | Pride