Acyl-CoA Quantification by LC-MS/HRMS

Acyl-CoA analyses were performed by liquid chromatograpy-mass spectrometry/high-resolution mass spectrometry (LC-MS/HRMS) as previously described (75 (link)). Briefly, approximately 20–50 ×10⁶ acinar cells were cultured in suspension in low adhesion 6 mm petri dishes. At harvest, cells were placed on ice, transferred to 15 ml falcon tubes and centrifuged at 600 xg for 2 minutes at 4 °C. Medium was aspirated and the cell pellet resuspended in 1 mL 10% (w/v) trichloroacetic acid (Sigma-Aldrich, catalog #T6399) for acyl-CoA extraction. For quantification, an equal amount (100 μL) of ¹³C₃¹⁵N₁-labeled acyl-CoA internal standard (75 (link)) was added to each sample. Samples were pulse sonicated, centrifugated and the supernatant was purified by solid-phase extraction using Oasis HLB 1cc (30 mg) SPE columns (Waters). Eluate was evaporated to dryness under nitrogen gas and re-suspended in 50 μL of 5 % 5- sulfosalicylic acid (w/v) for injection. Samples were analyzed by an Ultimate 3000 autosampler coupled to a Thermo Q-Exactive Plus instrument in positive electrospray ionization (ESI) mode. For isotopic tracer analysis, isotopic enrichment from [U-¹³C]glucose or [U-¹³C]palmitate or [U-¹³C]leucine was calculated to compensate for the non-linearity of isotopic enrichment using the FluxFix calculator (76 (link)).