ZDHHC_metabolomics_chemgen_2023.zip

MetabolomicsAcyl- and probe-Coenzyme A analysis.HEK293T cells were seeded in 6-well plates, grown to 70% confluency in media containing 0.5% FBS and treated with 30 mM YnPal or 18-Bz for 2 h. Cells were dislodged into their growth media by pipetting and pelleted by centrifugation (500 x g, 5 min). The cell pellet was washed twice by resuspending in ice-cold PBS and pelleting by centrifugation.Sample extractionTo each sample, 400 µL chloroform was added and vortexed for ~1 min, followed by addition of 200 µL methanol and a repeated vortex. Samples were incubated in a water bath sonicator (4°C, 1 h), with 3 × 8 min sonication pulses, followed by centrifugation (4°C, 10 min, 17,000 x g). The supernatant was transferred to a new Eppendorf 1.5 mL tube (E1). The pellet was re-extracted with 450 µL methanol:water (2:1 v:v, containing internal standard, 13C3-Malonyl-CoA), sonicated (8 min, 4°C) and centrifuged, as above. The supernatant was added to the first extract (E1). Combined extracts were dried using a speedvac concentrator, re-suspended in 350 µL chloroform:methanol:water (1:3:3, v/v), and centrifuged, as above. The upper, aqueous phase containing the polar metabolites (including probe, probe-CoA, and acyl-CoA molecules) was dried using the speedvac concentrator and resuspended in 100 µL acetonitrile/ammonium carbonate 20 mM (7:3, v/v) for LC-MS injection.Liquid chromatography-mass spectrometry (LC-MS)Chromatography conditions:Chromatography prior to all mass spectrometry was performed using an adaptation of a method previously described61. Samples were injected into a Dionex UltiMate 3000 LC system (Thermo Fisher) with a Phenomenex Luna C18(2) 100 Å (100 x 2 mm, 3 μm) column coupled with a SecurityGuard C18 guard column (4 x 2 mm). Analytes were separated using 20 mM ammonium carbonate in water (Optima HPLC grade, Sigma Aldrich) as solvent A and acetonitrile (Optima HPLC grade, Sigma Aldrich) as solvent B at 0.3 mL/min flow rate. Elution began at 5% Solvent B, maintained for 3 min, increased to 100% B over 12 min, followed by a 3 min wash of 100% B and subsequent 3 min re-equilibration to 5% B. Other parameters were as follows: column temperature, 30°C; injection volume, 10 μL; needle wash, 50% methanol; autosampler temperature, 4°C.High resolution mass spectrometryPost-chromatography, high resolution (HR) MS was performed with positive and negative polarity switching using a Q-Exactive Orbitrap (Thermo Fisher) with a HESI-II (Heated electrospray ionizati