Agilent 7890 5975c gc ms system

Cells were incubated in the culture medium supplemented with 5 mM ¹³C₆-glucose (Sigma–Aldrich, 389374) under the indicated conditions. Metabolites were extracted from cells as previously described (38 ). Briefly, each group of cells was collected and immediately flash frozen in liquid N₂, metabolites were extracted with ice-cold methanol, and lysates were centrifuged at 18,000g for 15 min to remove protein. The supernatant was dried in an evaporator and resuspended in 200 μl pyridine. Metabolites were further derivatized by addition of 50 μl MTBSTFA containing 1% t-BDMCS at 60 °C for 1 h. Samples were analyzed using Agilent 5MS column in the Agilent 7890/5975C GC/MS system (Agilent Technologies). Peaks representing each metabolite were extracted and integrated using MassHunter software (Agilent Technologies). ¹³C-labeled metabolite data were presented as percentage of ¹³C-labeled metabolites, which was calculated by dividing the labeled ions with total ion intensity. The labels were irrespective of carbon position. The distribution of mass isotopologs was corrected for natural abundance. We used IsoCor, a scientific software designed for the purpose of isotope labeling experiments, to correct the raw MS data for both all naturally occurring isotopes and purity of the isotopic tracer. The website for the software (IsoCor) is http://metasys.insa-toulouse.fr/software/isocor/.

Cells were collected in 80% (vol/vol) methanol, followed by repeated freezing and thawing. Insoluble material in the lysates was removed by centrifugation, and the supernatants were dried in a rotary evaporator and resuspended in 75 μl of pyridine. Metabolites were further derivatized by the addition of 25 μl of MTBSTFA containing 1% tert-butyl dimethyl chlorosilane at 60°C for 1 h. The samples were analysed using an Agilent DB-5MS column in an Agilent 7890/5975C GC/MS system (Agilent Technologies). Peaks representing each defined intensity were identified from known standards.
Formate was analysed by GC-MS as described previously (Lamarre et al, 2014 (link)). Briefly, cells were collected, and metabolites were extracted with ice-cold 80% (vol/vol) methanol, followed by centrifugation to remove insoluble material. The supernatants were dried in a rotary evaporator and resuspended in 50 μl of H₂O, and alkylation was carried out by mixing 20 μl of phosphate buffer with 130 μl of 100 mM pentafluorobenzyl bromide, vortexing for 1 min, and incubating the samples at 60°C for 15 min. Next, the tubes were cooled to room temperature, and 330 μl of n-hexane was added and vortexed for 1 min until the two layers were separated. A 200 μl volume of the top layer was transferred to glass inserts and analysed on a GC-MS system equipped with an Agilent HP-INNOWax GC column.