2.5. Extraction and analysis of acyl-CoAs by LC-MS

After 3 d fermentation, 10 mL of S. ablus J1074 broth was centrifuged at 3500×g for 10 min at 4 °C to collect the bacteria, then was frozen with liquid nitrogen and stored at −80 °C for subsequent extraction of acyl-CoAs. Three biological replicates were used. When extracting the acyl-CoAs, the sample was chilled in ice, then two zirconia glass beads (2 mm), 100 μL of zirconia glass beads (1 mm), and 1 mL of ice-cold monopotassium phosphate buffer (67 mM, pH 4.9) were added to the sample, and the bacteria were ground twice using a grinder (JXFSTPRP-24L, Jingxin, Shanghai, China, 60 Hz, stop for 10 s after 60 s) that was precooled at −40 °C. Next, 500 μL of precooled isopropanol was added, and the sample was ground again. After grinding, 1 mL of precooled acetonitrile and 60 μL of saturated ammonium sulfate solution (room temperature) was added, and the sample was ground once. After grinding, the tube was placed on ice for 10 min, centrifuged at 12,000×g at 4 °C for 10 min, the supernatant transferred into a new tube, lyophilized and then stored at −80 °C for subsequent analysis. The sample was resuspended in 200 μL of 50% methanol, vortexed for 1 min, and then water bath sonicated for 2 min. The sample was centrifuged at 12,000 ×g at 4 °C for 10 min and the supernatant used for acyl-CoA analysis using a HPLC-high resolution mass spectrometer (HPLC-HRMS, UltiMate 3000 system, Dionex, Sunnyvale, USA coupled with a Q Exactive Orbitrap mass analyzer, Thermo Fisher, Waltham, USA).

Chromatographic separation was achieved at 25 °C on a SeQuant ZIC-pHILIC column (150 × 2.1 mm, 5 μm; Merck, Darmstadt, Germany) coupled with a 20 mm SeQuant ZIC-pHILIC guard column at a flow rate of 0.2 mL/min. A linear gradient of solvent A (5% acetonitrile, 95% 15 mm NH₄HCO₃ pH 8.5) and solvent B (acetonitrile) was performed as follows: 0–1 min at 90% B, 1–13 min 90%–30% B, 13–16 min 30% B, 16–17 min 30%–90% B, followed by 8 min of re-equilibration at 90% B. The injection volume was 1 μL and the autosampler was set at 4 °C during the analysis. The Q Exactive mass spectrometer was operated in electrospray ionization (ESI) negative mode. Source parameters were optimized with a spray voltage of 3.2 kV (−). The other parameters were set as follows: capillary temperature, 320 °C; auxiliary gas temperature, 300 °C; sheath gas, 40 Arb; auxiliary gas, 10 Arb; sweep gas, 0 Arb, and the S-lens RF level was set at 50. The Q Exactive detector was operated in full scan mode plus data-dependent MS² (dd MS²) mode. In the full scan mode, the resolution was set at 70,000. The automatic gain control (AGC) target and maximum injection time (IT) were set at 1 × 10⁶ ion capacity and 100 ms, respectively. In data-dependent MS² (dd MS²) mode, the resolution was set to 17,500. The AGC target and maximum IT were set at 2 × 10⁵ ion capacity and 50 ms, respectively. The inclusion list was on. All targeted metabolites m/z at [M − H]^- were included in the list and prepared to fragment. The scan range was set at m/z 100–1500. The normalized collision energies (NCE) were 20%, 40%, and 60%. The isolation window was set at 1.2 Da. The apex trigger was set at 5–15 s, the loop count was set at 3, and the dynamic exclusion was set at 5 s.

The standard solutions were prepared at six individual calibration concentrations between 0.1 and 5 mg/L for acetyl-CoA and propionyl-CoA; or 0.2–5 mg/L for malonyl-CoA and methylmalonyl-CoA.