Biofluid and Tissue Metabolomics Analysis

The biofluid metabolomics was examined using an Agilent 7890 gas chromatography system equipped with a Pegasus 4D time of flight mass spectrometer (LECO, St. Joseph, MI, USA) [6 (link)]. The tissue metabolomics procedures were performed as follows. First, 100 mg of MG tissue from each sample was added to a 2-mL Eppendorf tube with 0.4 mL of methanol-chloroform (V_methanol: V_chloroform = 3:1) and 30 μL of L-2-chlorophenylalanine (1 mg/mL, stored in dH₂O) and was mixed by vortexing for 10 s. Second, steel balls were placed in the tube and milled for 5 min at 55 Hz. The sample was then centrifuged at 4 °C at 12,000 rpm for 15 min. Third, approximately 0.4 mL of supernatant was transferred into a 2 mL silylated vial. An equal volume (10 μL) of each sample was placed in a new 2 mL silylated vial as a mixed sample for the quality control of the stability of the equipment system, the standard deviation of the beginning, middle and ending retention time of the mixed samples was less than 0.2, which indicates good stability.