2.10 Metabolomics analysis

The samples from the seven tissues were harvested as previously described. For each sample, 20 mg of powder was prepared and further extracted with 400 µL of 80% aqueous methanol at 4 ℃, followed by centrifugation for 10 min at 12,000 rpm. LC-MS analysis was performed using the Waters Acquity UPLC System connected to an AB SCIEX 5500 QQQ-MS.
Gradient elution was achieved on a Waters Acquity UPLC BEH C18 column (100mm*2.1mm, 1.7µm) with water containing 0.1% formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.30 mL/min (X. Yu et al., 2020). The column temperature was maintained at 40℃. The gradient elution program was as follows: 1-10%B (0-1min),11-60%B (2-5min), 60-90%B (5-7min), held at 99 %B (7-9min), and allowed to equilibrate for a further 3min before the next injection and the last 8min of the chromatogram solutions were discarded. The injection volume was 4µL. MS data were recorded with the following parameters: Ion source, ESI; IonSource temperature, 450℃; IonSource Gas1, 55arb; IonSource Gas2, 55arb; IonSpray voltage, 4500V; Curtain Gas, 35arb; Collision GAS, 7arb.
Components eluting from the UPLC-QQQ-MS system were processed in MultiQuant for data preprocessing with default settings, except that each sample was normalized to the internal standard (X. Yu et al., 2020). After filtering for outliers, the data were used for the subsequent statistical analysis.