D4 succinate

Metabolite extraction and quenching were performed on plate with 1.5 mL ice cold extraction medium (90% methanol, 10% water) containing 1 µg/mL ribitol (A5502, Sigma Aldrich), phenyl β-d-glucopyranoside (292710, Sigma Aldrich), isoguanosine (sc-207768, Santa Cruz, Dallas, TX, USA), d4-succinate (293075, Sigma Aldrich) and methyl-tyrosine (M8131, Sigma Aldrich) as the internal standard. Cells were detached on ice by using a cell scraper, transferred into screw-cap tubes prefilled with 300 mg glass beads (G4649, Sigma Aldrich) and immediately frozen in liquid nitrogen until homogenization. Cells were homogenized using a Precellys tissue homogenizer (P000669-PR240-A, Bertin instruments, Montigny-le-Bretonneux, France) at −10 °C. Three cycles of homogenization for 15 s at 6500 rpm were applied with 10-s breaks in between cycles. Samples were then centrifuged (20,000× g) for 10 min at 4 °C to remove cell debris and protein precipitates, and 500 µL of each supernatant was transferred into two new reaction tubes for GC-MS and LC-MS analyses. Finally, extracts were dried using a vacuum rotator (Eppendorf, Hamburg, Germany) and flushed with nitrogen. The DNA content in the extracts was measured using NanoDrop 1000 (Thermo Fisher Scientific).

Metabolites were extracted by adding 810 µL of ice-cold methanol (VWR, Rednor, Pennsylvania, USA), containing 10 µmol L⁻¹ ethylparaben (Fluka, Buchs, Switzerland), 2 µmol L⁻¹ 3-nitro-L-tyrosine (Sigma-Aldrich, St. Louis, MO, USA), and 4 µmol L⁻¹ d₄- succinate (Sigma-Aldrich) to 90 µL of plasma. The internal standards ethylparaben, 3-nitro-L-tyrosine and d₄-succinate were used for quality control for the assessment of the system stability. Proteins were pelleted in a centrifuge (HERMLE, Wehingen, Germany) at 18,620 rpm for 10 min at 4 °C. The supernatant was evaporated to dryness in a vacuum concentrator (Eppendorf, Hamburg, Germany) at room temperature, followed by resuspension in 90 µL 50% methanol.