Metabolite profiling by LC-MS

LC–MS-based metabolite profiling was performed on an Accela system coupled to an Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK) operating with electrospray ionisation (ESI) running in the negative (ESI−) and positive (ESI+) modes as previously described in (Kim et al. 2015 (link)). Briefly, the spray voltage was 4500 V (ESI+) and 3500 V (ESI−), capillary voltage was 40 V (ESI+) and 30 V (ESI−), tube lens voltage was 70 V for the both modes and skimmer voltage was 20 V (ESI+) and 18 V (ESI−). The temperature for capillary and probe was maintained at 275 °C and 150 °C, respectively. Chromatographic separation was carried out using ZIC-pHILIC (4.6 × 150 mm and 5 μm particle size, Merck Sequant). The mobile phase composed of 20 mM ammonium carbonate in water (solvent A) and 100% acetonitrile (solvent B). Metabolites were separated according to a linear gradient as following: 0–15 min (20% A), 15–17 min (95% A), and 17–24 min (20% A) at 300 µl/min flow rate. The injection volume of 10 µl and the column was kept at 45 °C.