293T cells transfected with IDH2 wild-type (A) or IDH2 R172K (B) were provided fresh culture medium the day following transfection. 24 hours later, the medium was collected, from which organic acids were extracted, purified, and derivatized with MTBSTFA. Shown are representative gas chromatographs for the derivatized organic acids eluting between 30 to 34 minutes, including aspartate (Asp) and glutamate (Glu). The arrows indicate the expected elution time of 32.5 minutes for MTBSTFA-derivatized 2HG, based on similar derivatization of a commercial R(-)-2HG standard. Metabolite abundance refers to GC-MS signal intensity. (C) Mass spectrum of the metabolite peak eluting at 32.5 minutes in (B), confirming its identity as MTBSTFA-derivatized 2HG. The structure of this derivative is shown in the inset, with the tert-butyl dimethylsilyl groups added during derivatization highlighted in green. m/e− indicates the mass (in atomic mass units) to charge ratio for fragments generated by electron impact ionization (D) Cells were transfected as in (A) and (B), and after 48 hours intracellular metabolites were extracted, purified, MTBSTFA-derivatized, and analyzed by GC-MS. Shown is the quantitation of 2HG signal intensity relative to glutamate for a representative experiment. See also .