The UltiMate 3000 Ultra High-performance Liquid Chromatograph (Thermo Fisher, USA) and Thermo Q-Exactive Orbitrap Mass Spectrometer were used for ultrapure LC-MS/MS analysis (Thermo Fisher). Chromatographic separations were performed on Waters ACQUITY UPLC HSS T3 (100 mm × 2.1 mm × 1.8 μm). Thereafter, all the raw data were fed into Progenesis QI (Waters, Milford, MA, USA) and SIMCA-P14.0 (Umetrics AB, Umea, Vasterbotten, Sweden), software for further analysis [15 (link)]. The partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) supervised pattern recognition methods were applied to identify the overall metabolic differences among the three groups and the variable importance in the projection (VIP) was used to identify characteristic metabolites in the three groups. Distinct metabolites were analyzed based on VIP > 1.0 and P < 0.05. Further information is provided in the Supplemental Materials and Methods.
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