2.7 Metabolite sample pretreatment and MS-based analytical procedures
The above-mentioned sample extracts were evaporated to dryness using Rap-Vap (Labconco, Kansas City, MO). The obtained residue was redissolved in deionized water to obtain a final weight of 600 µg. Subsequently, the extracts were split and stored at -80℃ until analysis.
Samples for amino acids quantification were analyzed using GC-MS (7890A GC, Agilent, Santa Clara, CA, USA) instrument coupled to a 5975C MSD single quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). Briefly, 100 µL cell extracts were freeze-dried and derivatized by adding 75 μL acetonitrile and 75 μL N-methyl-N- (tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) at 70°C for 60 min. Then the derivatized sample was cooled to room temperature and centrifuged at 12000×g for 2 min. The supernatant was collected for further analysis.
Samples for organic acids, sugar phosphates, adenosine nucleotides and coenzymes were analyzed using LC-MS/MS (Dionex Ultimate 3000 UPLC system coupled to TSQ Quantum Ultra mass spectrometer, Thermo Scientific). The absolute quantification of these metabolites was achieved using GC-IDMS and LC-MS/MS-IDMS methods [42]. The details of the analytical procedure have been described elsewhere [22].