LC-HRMS was performed in an Accela 600 LC system (Thermo Fisher Scientific) coupled to an Exactive (Orbitrap) mass spectrometer (Thermo Fisher Scientific). Metabolite separation was done using a SeQuant ZIC-pHILIC column (4.6 mm × 150 mm, 5 μm) (Merck). The mobile phase consisted of an aqueous solution of ammonium carbonate (20 mM, pH 9.2) (A) and acetonitrile (B) and the following gradient profile was applied: linear increase of A from 20 to 80% at 0 to 30 min, 92% A 31–37 min and linear decrease of A to 20% at 37–46 min. Total injection volume was 20 µL and samples were maintained at 4 °C throughout analysis. The spectrometer was operated in both positive and negative electrospray ionisation (ESI) modes, full-scan mode over a mass range of m/z 70–1200 at a resolution of 50,000. The capillary temperature was 320 °C and the seath and auxiliary gas flow rates were 50 and 17 units, respectively. Thermo raw files were converted to mzML files using ProteoWizard, separated into ESI positive and negative modes, and imported to MZMine 2.53 for peak processing. Metabolite identification was performed using an in-house standard library and peak areas were exported for analysis. The (peak area) energy charge ratio (ECR) was calculated from the formula ECR = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]), where [metabolite] is the metabolite’s peak area [19 (link)].
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