UHPLC-MS/MS Metabolite Profiling Protocol

The UHPLC-MS/MS measurement was performed using Ultimate 3000 liquid chromatography coupled with Q-Exactive Orbitrap HRMS (Thermo Fisher Scientific, Waltham, MA, USA) based on the previously described method [26 (link)]. A chromatographic separation was performed on a Thermo Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 µm) maintained at 40 °C, with a flow rate of 0.4 mL/min. The samples were kept under 4 °C and injection volume was 5 µL. The gradient elution was composed of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) and performed for 22 min, as shown in Supplementary Table S1. The Q-Exactive Orbitrap HRMS was equipped with heated electrospray ionization (HESI) source using the following source parameters: sheath gas flow—55 arb (arbitrary units); aux gas flow—10 arb; spray voltage—+4.0/−3.5 KV; capillary temperature—350 °C; S-lens RF level—55; aux gas heater temperature—300 °C. For MS, resolution was set as 70,000 and Scan range was 70 to 1000. For MS/MS, data-dependent acquisition (DDA) mode was used, and the top 10 precursors were fragmented by 15, 30, and 45 collision energy values.