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
Date made available | 2021 |
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Publisher | PRoteomics IDEntifications Database |
Date of data production | 15 Jun 2021 |
Keywords
- Human
- senescent
- heat
- shock
- MSC
- mBBr
- mesenchymal
- monobromobimane
- stem cell
Equipment
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Biological Mass Spectrometry (BioMS) Facility
Knight, D. (Platform Lead), Warwood, S. (Senior Technical Specialist), Selley, J. (Technical Specialist), Taylor, G. (Technical Specialist), Fullwood, P. (Technical Specialist), Keevill, E.-J. (Senior Technician) & Allsey, J. (Technician)
FBMH Platform Sciences, Enabling Technologies & InfrastructureFacility/equipment: Facility