5975c inert ms system

Proline was measured with gas chromatography–mass spectrometry. For this, the samples were extracted and derivatized as previously described (23 (link), 54 (link)). In brief, polar metabolites were derivatized at 37°C with 20 μl of methoxyamine (20 mg/ml) in pyridine for an hour and half. Next, 15 μl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide, with 1% tert-butyldimethylchlorosilane was added to 7.5 μl of each sample and incubated for 1 hour at 60°C. Isotopolog distributions and metabolite concentrations were measured with a 7890 A gas chromatography (GC) system combined with a 5975C Inert MS system (Agilent Technologies). One microliter of sample was injected into a DB35MS column in splitless mode using an inlet temperature of 270°C. The carrier gas was helium with a flow rate of 1 ml/min. Upon injection, the GC oven was set at 100°C for 1 min and then increased to 105° at 2.5°C/min and with a gradient of 2.5°C/min lastly to 320° at 22°C/min. Isotopolog distributions and peak areas were extracted from the raw ion chromatograms using an in-house Matlab script, which applies consistent integration bounds and baseline correction to each ion. In addition, we corrected for naturally occurring isotopes and normalized peak areas to the protein content of the sample and internal standard glutaric acid.