Targeted Metabolite Analysis by LC-MS

i) Sample quenching, extraction, and preparation: All chemicals used for LC-MS metabolomic analyses were obtained from Sigma-Aldrich (Taufkirchen, Germany). Cells were collected by centrifugation at 8,000 × g for 8 min at room temperature (Eppendorf 5430R, Hamburg, Germany). The cell samples were quenched and extracted rapidly with 900 μL of 80:20 MeOH/H₂O (−80°C) and then frozen in liquid nitrogen. The samples were then frozen-thawed three times to release metabolites from the cells. The supernatant was collected after centrifugation at 15,000 × g for 5 min at −4°C and then stored at −80°C. The remaining cell pellets were re-suspended in 500 μL of 80:20 MeOH/H₂O (−80°C) and the above extraction process was repeated. The supernatant from the second extraction was pooled with that from the first extraction and stored at −80°C until LC-MS analysis [27 (link)]; ii) LC-MS analysis: The chromatographic separation was achieved with a SYnergi Hydro-RP (C18) 150 mm × 2.0 mm I.D., 4 μm 80 Å particles column (Phenomenex, Torrance, CA, USA) at 40°C. Mobile phase A (MPA) is an aqueous 10 mM tributylamine solution with pH 4.95 adjusted with acetic acid and Mobile phase B (MPB) is 100% methanol of HPLC grade (Darmstadt, Germany). The optimized gradient profile was determined as follows: 0 min (0% B), 8 min (35% B), 18 min (35% B), 24 min (90% B), 28 min (90% B), 30 min (50% B), 31 min (0% B). A 14-minute post-time equilibration was employed, bringing total run-time to 45 min. Flow rate was set as a constant 0.2 mL/min [57 (link)]. LC-MS analysis was conducted on an Agilent 1260 series binary HPLC system (Agilent Technologies, Waldbronn, Germany) coupled to an Agilent 6410 triple quadrupole mass analyser equipped with an electrospray ionization (ESI) source. Injected sample volume for all cases was 10 μL; capillary voltage was 4000 V; and nebulizer gas flow rate and pressure were 10 L/min and 50 psi, respectively. Nitrogen nebulizer gas temperature was 300°C. The MS was operated in negative mode for multiple reaction monitoring (MRM) development, method optimization, and sample analysis. Data were acquired using Agilent Mass Hunter workstation LC/QQQ acquisition software (version B.04.01) and chromatographic peaks were subsequently integrated via Agilent Qualitative Analysis software (version B.04.00); iii) Targeted metabolite analysis: a total of 24 metabolites were selected for LC-MS based targeted metabolite analysis in this study. The abbreviations, molecular weights and MRM values determined and optimized for each of the 24 detected metabolites as well as the product ion formulas were provided in Additional file 1: Table S1. The standard compounds for these 24 metabolites were purchased from Sigma, and their MS and MS/MS experimental parameters were optimized with the mix standard solution. All metabolomics profile data was first normalized by the internal control and the cell numbers of the samples, and then subjected to Principal Component Analysis using software SIMCA-P 11.5 [58 (link)].