Acetyl-CoA sample preparation and LC-MS/MS analysis

Acetyl-CoA was extracted as previously described⁵⁸. Briefly, cold methanol (500 μl; −20 °C) was added to the cell pellets, and the mixture was shaken for 30 s (10 °C, 2000 r.p.m., Thermomixer C, Eppendorf). Cold chloroform (500 μl; −20 °C) was added, the mixture was shaken for 30 s, and 200 μl of water (4 °C) added afterwards. After 30 s shaking and 10 min on ice, the mixture was centrifuged (21,000 × g, 4 °C, 10 min). The upper layer was collected and evaporated. The dry residue was re-dissolved in eluent buffer (500 μl) and centrifuged (21,000 × g, 4 °C, 10 min) before placing in LC-MS vials. Acetyl CoA was analyzed as previously described⁵⁹. Briefly, the LC-MS/MS instrument consisted of an Acquity I-class UPLC system and Xevo TQ-S triple quadrupole mass spectrometer (both Waters) equipped with an electrospray ion source. LC was performed using a 100 × 2.1-mm i.d., 1.7-μm UPLC Kinetex XB-C18 column (Phenomenex) with mobile phases A (10 mM ammonium acetate and 5 mM ammonium hydrocarbonate buffer, pH 7.0, adjusted with 10% acetic acid) and B (acetonitrile) at a flow rate of 0.3 mL min-1 and column temperature of 25 °C. The gradient was set as follows: 0–5.5 min, linear increase 0–25% B, then 5.5–6.0 min, linear increase till 100% B, 6.0–7.0 min, hold at 100% B, 7.0–7.5 min, back to 0% B, and equilibration at 0% B for 2.5 min. Samples kept at 4 °C were automatically injected in a volume of 5 μl. Mass spectrometry was performed in positive ion mode, monitoring the MS/MS transitions m/z 810.02 → 428.04 and 810.02 → 303.13 for acetyl-CoA. Spikes of defined amounts of AcCoA were added to the samples to confirm the absence of signal inhibition (matrix effect) in the analyzed extracts. Quantification of AcCoA was done against an external calibration curve with 1–1000 ng mL–1 range of AcCoA concentrations using TargetLynx software (Waters).