Stable iotope enrichment analysis using 13C515N2-glutamine

MEFs were grown and extracted as described in the whole-cell metabolite analysis section. Cells were washed twice with 1 ml of Dulbecco’s PBS (Gibco, catalogue no. 14190250) before adding the tracing buffer (DMEM + 10% dialysed FBS, 25 mM glucose, 1 mM sodium pyruvate and 2 mM l-glutamine-¹³C₅¹⁵N₂ (Sigma, catalogue no. 607983)).

Metabolic analysis was performed as described previously³¹ using an Acquity iClass UPLC (Waters) equipped with a ZIC-pHILIC 2.1 × 150 mm (5-μm particle size) column (Sequant) coupled to an Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The HPLC flow rate and gradient settings as well as MS settings were identical to those reported previously³¹.

Data analysis was performed using TraceFinder software (Thermo Fisher Scientific). The identity of each compound was validated by reference compounds and peak areas of selected ¹³C ¹⁵N or ¹³C¹⁵N isotopes of each analysed compound was extracted from either [M – H⁺]⁻ (nucleotides, α-ketoglutarate, succinate, fumarate, malate and citrate) or [M + H⁺]⁺ (glutamine, glutamate and aspartate) ions. For the differential isotope enrichment analysis, the sum of all peak areas of the extracted isotopes of a compound was calculated together with the fraction of the individual isotope. In addition, we calculated the atom fraction enrichment factor, which describes the relative contribution of stable isotopes to the total area of each compound. Isotope peaks were extracted using a mass accuracy (<5 ppm) and a retention time tolerance of <0.1 min. A list of analysed isotopologues and the individual metabolite pool sizes according to the sum of all isotopes are presented in Supplementary Table 1.