Uplc q tof ms

UPLC-Q-TOF-MS (1290 UPLC-6540, Agilent Technologies Inc., United States) was used to analyze the prepared samples. Chromatographic conditions were as follows: An Agilent ZORBAX Eclipse Plus C18 (3.0 × 100 mm, 1.8 μm) column was used and the mobile phase system was composed of acetonitrile (A) and water (contained 0.1% formic acid). A gradient elution procedure was used, as follows: 0–10 min, 5–15% A; 10–15 min, 15–20% A; 15–25 min, 25–45% A; 25–40 min, 45–80%; flow velocity, 0.4 ml/min; and the sample volume, 1 μl. Mass spectrometry testing conditions were as follows: ionization mode, electrospray ionization and the accurate mass data correction using electrospray ionization-L Low Concentration Tuning Mix (G1969-85000). Positive and negative ion analysis mode and MRE scan mode were adopted. The range of full-mass scanning was 100–1700 m/z. Sheath gas temperature was 350°C and the capillary voltage of 4.0 KV. The desolventize gas was nitrogen, and the temperature of the dry gas was 325°C, with a flow rate of 6.8 L/min. Secondary mass spectrometry was performed by dependent scanning, and the first three strengths were selected for collision induced dissociation (CID), based on of primary scanning to obtain secondary mass spectrometry data. The secondary fragment scan range was 50–1000 and fragment voltages were 10, 20, and 30 KV.

Metabolites were extracted with 300 μL methanol and ultrasonic oscillation for 10 min. The supernatant was collected after centrifugation at 12000 rpm for 10 min at 4 °C. UPLC-QTOF/MS (Agilent Technologies, USA) was implemented for metabolites detection with six technical replicates. The experimental quality was evaluated by quality control (QC) samples. Features alignment, picking, and identification were performed by Progenesis QI (Waters, Nonlinear Dynamics, Newcastle, UK). By combining the univariate and multivariate statistical analysis, significantly changed features (P value< 0.05, VIP > 1) were acquired. Those features were further annotated by Progenesis QI with public databases including Metlin, LIPID MAPS, PubChem, YMDB (Yeast Metabolome Database) and KEGG.