D8 valine

Intracellular metabolites were obtained after washing cells with 2 volumes of ice-cold PBS and floating on liquid nitrogen. Plates were stored at –80 °C until extraction. Metabolites were extracted with 1 mL 80% MeOH pre-cooled to –80 °C containing 10 nmol [D₈]-valine as an internal standard (Cambridge Isotope Labs). Insoluble material was removed by centrifugation at 21,000×g for 15 min at 4 °C. The supernatant was evaporated to dryness at 42 °C using a SpeedVac concentrator (Thermo Savant). Samples were resuspended in 35 μL LC-MS-grade water prior to analysis.

Extraction buffer was prepared by adding 2:2:1 methanol:acetonitrile:water to internal standards at 1 μg/mL each (D4-Citric Acid, ¹³C5-Glutamine, ¹³C5-Glutamic Acid, ¹³C6-Lysine, ¹³C5-Methionine, ¹³C3-Serine, D4-Succinic Acid, ¹³C11-Tryptophan, and D8-Valine; Cambridge Isotope Laboratories). Plasma volume was included in the calculations for the water portion of the buffer. A total of 720 µL of extraction buffer was then added to 40 μL of each plasma sample. Samples were placed on a rotating platform at –20 °C for 1 h and centrifuged at 4 °C for 10 min at 21,000× g. A total of 300 µL of the metabolite extracts was transferred into fresh microcentrifuge tubes. An equal volume of each extract was pooled to serve as a quality control (QC) sample, which was analyzed at the beginning, end, and after every tenth sample throughout the instrument run. Extraction buffer alone was analyzed as a processing blank sample to account for carryover or background contamination from the sample extraction process. Metabolite extracts, the QC sample, and the processing blank were evaporated to dryness using a speed-vacuum. All samples were reconstituted in 30 μL of acetonitrile/water (1:1, v/v), vortexed for 10 min, and incubated at –20 °C for 18 h. Samples were then centrifuged at 4 °C for 2 min at 21,000× g and the supernatant was transferred into autosampler vials for analysis.

For metabolite extraction, samples were extracted in ice cold 2:2:1 methanol/acetonitrile/water which contained 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.  The ratio of extraction solvent to sample volume was 18:1. Tissue samples were lyophilised overnight prior to extraction. Tissues were homogenised using a ceramic bead mill homogeniser, after the addition of extraction buffer. Samples were then incubated at −20°C for 1 h followed by a 10 min centrifugation at maximum speed. Four hundred microliters of supernatants were transferred to fresh tubes.  Pooled QC samples were prepared by adding an equal volume of each sample to a fresh 1.5 ml microcentrifuge tube. Processing blanks were utilised by adding extraction solvent to microcentrifuge tubes. Samples, pooled QCs, and processing blanks were evaporated using a speed‐vac. The resulting dried extracts were reconstituted in 40 μl of acetonitrile/water (1:1, V/V), vortexed and samples, blanks, and pooled QCs were then analysed using liquid chromatographymass spectrometry (LC–MS).