LC-MS metabolomics analysis and metabolites identification

The LC-MS analysis was performed on hydrophilic interaction chromatography coupled with electrospray ionization to the Q Exactive PLUS hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific) as previously described [86]. The LC separation was performed on a XBridge BEH Amide column (150 mm × 2.1 mm, 2.5 μm particle size, Waters, Milford, MA) using a gradient of solvent A (95%/5% H₂O/ acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide, pH 9.4), and solvent B (20%/80% H₂O/ acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide, pH 9.4). The gradient was 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; 22 min, 100% B. The flow rate was 300 μL/min. Injection volume was 5 μL and column temperature 25 °C. Each sample was analyzed twice in both negative and positive ionization mode with a resolution of 70,000 at m/z 200. The automatic gain control target was 3 × 10⁶. The maximum injection time was 50 ms. Scan range was 75–1000. The MS2 spectra were collected from pooled samples under ddMS2 mode. The targeted metabolite data analysis was performed in MAVEN [87]. The compound identification was based on the accurate mass and the retention time learned from in-house chemical collection which includes 344 metabolites. The untargeted analysis was performed in Compound Discoverer (Thermo Scientific).