Targeted and Untargeted Metabolomics Analysis

Metabolomics analysis was performed as previously described [70 (link)]. Briefly, the cells were washed twice with cold PBS and incubated with pre-chilled 80% methanol (−80 °C) for 1 h at −80 °C. Then, the cells were scraped in 80% methanol on dry ice and centrifuged for 5 min. The extracted metabolites in the supernatant were concentrated completely to dryness with a lyophilizer and the protein concentrations of centrifuged pellets were used for normalization. The dried metabolites were dissolved in 80% methanol and analyzed by LC–MS/MS. We used the TSQ Quantiva™ Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with positive/negative ion switching for targeted metabolites quantitative analysis. The Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was chosen for untargeted metabolites profiling. Metabolites were identified based on the retention time and the accurate mass measured with <5 ppm mass accuracy. Quantitative information of metabolites was analyzed by TraceFinder.