Acetyl CoA Measurement

Intracellular acetyl-CoA levels were measured using mass spectrometry. Cells were grown to mid-logarithmic phase (OD₆₂₀ = 0.4) in C+Y. Cells were pelleted at 6,000 x g for 5 min at room temperature and then lysed using methanol/ 2% acetic acid and subjected to chloroform treatment. 250 pmol of [¹³C₂] acetyl-CoA (Sigma) was added. Metabolites were extracted from the aqueous phase via a 2-(2-pyridyl) ethyl column, eluted using 95% ethanol/ 50mM ammonium formate, and blown under nitrogen for analysis. Samples were resuspended in 90% methanol + 15mM ammonium hydroxide. Mass spectrometry of acetyl-CoA was performed using a Finnigan TSQ Quantum (Thermo Electron) triple-quadrupole mass spectrometer. The instrument was operated in positive mode using single ion monitoring (SIM) neutral loss scanning corresponding to the loss of the phosphoadenosine diphosphate from CoA species. The ion source parameters were as follows: spray voltage, 4,000 V; capillary temperature, 250°C; capillary offset, −35 V; sheath gas pressure, 10; auxiliary gas pressure, 5; tube lens offset was set by infusion of the polytyrosine tuning and calibration in electrospray mode. Acquisition parameters were as follows: scan time, 0.5 s; collision energy, 30 V; peak width Q1 and Q3, 0.7 FWHM; Q2 CID gas, 0.5 mTorr; source CID, 10 V; neutral loss, 507.0 m/z; SIM mass of 810 m/z with a scan width of 8 m/z to capture the signal from light and heavy acetyl-CoA. Acetyl-CoA levels were compared using unpaired parametric t test in Prism 6.