Metabolomics Profiling by LC-MS/MS

The methods and steps referred to the papers of other scholars (Xie et al., 2019 (link); Wang et al., 2019a (link),b (link); Ren et al., 2022 (link)). The metabolites were extracted using a 400 μL methanol: water (4,1, v/v) solution with 0.02 mg/mL L-2-chlorophenylalanin as internal standard. After protein precipitation and centrifugation, the supernatant was carefully transferred to sample vials for LC–MS/MS analysis. A pooled quality control sample (QC) was prepared by mixing equal volumes of all samples. The QC samples were disposed and tested in the same manner as the analytic samples. The instrument platform for this LC–MS analysis is UHPLC-Q Exactive HF-X system of Thermo Fisher Scientific. After the mass spectrometry detection is completed, the raw data of LC/MS is preprocessed by Progenesis QI (Waters Corporation, Milford, United States) software. At the same time, the metabolites were searched, identified and analyzed.