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].