Metabolite Profiling by LC-MS/MS

LC–MS data were acquired using an Ultimate 3000 DGP-3600RS HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA)^{3 (link),12 (link)}. Briefly, LC separation was performed on a ZIC-pHILIC column (Merck SeQuant, Umea, Sweden; 150 mm × 2.1 mm, 5 μm particle size). Acetonitrile (A) and 10 mM ammonium carbonate buffer, pH 9.3 (B) were used as the mobile phase, with a linear gradient from 80–20% A in 30 min at a flow rate of 100 μL/min. The mass spectrometer was operated in full-scan mode with a 100–1000 m/z scan rate and automatic data-dependent MS/MS fragmentation scans. For each metabolite, we chose a singly charged, [M+H]⁺ or [M−H]⁻, peak (Supplementary Table S2). Peak detection and identification of metabolites were performed using MZmine 2 software (http://mzmine.github.io/)^{26 (link)}. Detailed data analytical procedures and parameters have been described previously^{25 (link)}. Metabolite peaks were identified by comparing their m/z values and retention times with pure standards listed in previous reports^{3 (link),24 (link),25 (link),56 (link)}.