Acyl- 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.
To 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 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 spectrometry
Post-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 ionization) probe. MS parameters were as follows: spray voltage, 3.5 kV and 3.2 kV (for positive and negative modes, respectively); probe temperature, 320°C; sheath and auxiliary gases, 30 and 5 arbitrary units (au), respectively; full scan range: 100 to 1300 m/z with settings of AGC target and resolution as Balanced and High (3 × 106 and 70,000), respectively. Data were recorded using Xcalibur 3.0.63 software (Thermo Fisher). Mass calibration was performed for both ESI polarities before analysis using the standard Thermo Fisher Calmix solution. Qualitative analysis was performed using Xcalibur FreeStyle 1.8 SP1 and Tracefinder 5.1 software (Thermo Fisher) according to the manufacturer’s workflows. Masses, retention times, and fragmentation of all relevant sample-derived molecules were compared to authentic chemical standards.
MS parameters were optimized by direct infusion of 16 μM acyl-CoAs dissolved in 10 mM MeOH/ammonium acetate at 5 μL/min into an TSQ Quantiva triple quadrupole MS (Thermo Fisher). The heated electrospray was set in positive mode with the following parameters: capillary voltage, 3472 V; sheath gas, 60 au; aux gas, 10 au; sweep gas, 1 au; ion transfer tube temp, 325°C; vaporizer temp, 275°C. A selected reaction monitoring (SRM) function was applied for the simultaneous detection of acyl-CoA and probe-CoA molecules with RF lens and collision energies as shown in the Supplementary Table 7. Data were recorded using the Xcalibur 126.96.36.199 software and analysed using QuanBrowser 4.5.445.18 (Thermo Fisher).
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