Cells are lyophilized prior to extraction with 1 mL of ice-cold 2:2:1 methanol/acetonitrile/water containing a mixture of 9 internal standards (d4-Citric Acid, 13C5-Glutamine, 13C5-Glutamic Acid, 13C6-Lysine, 13C5-Methionine, 13C3-Serine, d4-Succinic Acid, 13C11-Tryptophan, d8-Valine; Cambridge Isotope Laboratories) at a concentration of 1 µg/ml each. Samples are scraped from the plate and then frozen in liquid nitrogen followed by a 10-minute sonication and incubated while gently rotating at –20 °C for 1 hour. After incubating, the samples are centrifuged for 10-minutes at maximum speed (15,000g). The supernatants are then transferred to fresh 1.5 ml microcentrifuge tubes. A pooled quality control (QC) sample is prepared by adding an equal volume of each sample to a fresh 1.5 ml microcentrifuge tube. Processing blanks are utilized by adding extraction solvent to microcentrifuge tubes. Then a 100µL aliquot of all samples, pooled QCs, and processing blanks are evaporated in 1.5 ml microcentrifuge tubes using a speed-vac at room temperature.
Dried extracts are reconstituted in 50 µL acetonitrile/water (1:1 v/v), vortexed well, and incubated in a -20 °C freezer overnight. In the morning, the resuspended samples are vortexed and centrifuged for 10 minutes at maximum speed, and the supernatant is transferred to LC-MS autosampler vials for analysis.
LC-MS data are acquired on a Thermo Q Exactive hybrid quadrupole Orbitrap mass spectrometer with a Vanquish Flex UHPLC system or Vanquish Horizon UHPLC system. The LC column used is a Millipore SeQuant ZIC-pHILIC (2.1 X 150 mm, 5 µm particle size) with a ZIC-pHILIC guard column (20 x 2.1 mm). The injection volume is 8 µL. Two mobile phases are used, solvent “A” containing 20 mM ammonium carbonate [(NH4)2CO3] and 0.1% Ammonium Hydroxide (v/v) [NH4OH [Note: pH is ~9.1] and solvent “B” containing 100% Acetonitrile. The method is run at a flow rate of 0.150 mL/min.
The gradient started at 80% B and decreased to 20% B over 20 minutes before returning to 80% B in 0.5 minutes and held there for 7 minutes. (PMID: 28388410). Note: there is a 2-minute pre-equilibration time prior to sample injection and during re-equilibration from 24.5 to 26.5 minutes flow is increased to 0.3 ml/min. The pooled QC samples are injected at the beginning, end, and every 8 samples throughout the run.
The mass spectrometer is operated in full-scan, polarity-switching mode from 1 to 20 minutes, with the spray voltage set to 3.0 kV, the heated capillary held at 275 °C, and the HESI probe held at 350 °C. The sheath gas flow is set to 40 units, the auxiliary gas flow is set to 15 units, and the sweep gas flow is set to 1 unit. MS data acquisition is performed in a range of m/z 70–1,000, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time at 200 ms. (PMID: 28388410)
Acquired LC-MS data are processed by Thermo Scientific TraceFinder 5.2 software, and metabolites are identified based on the University of Iowa Metabolomics Core facility standard-confirmed, inhouse library. NOREVA is used for signal drift correction using the pooled QC sample injections (PMID: 28525573). The data are normalized to the sum of all the measured metabolite ions in that sample.