Comprehensive 13C Metabolite Analysis

Polar fractions were reconstituted in 30 μl nanopure water, and analyzed on a Dionex ICS-5000+ion chromatograph interfaced to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operating at a resolution setting of 500,000 (FWHM at m/z 200) on MS1 acquisition to capture any and all ¹³C isotopologues^{40 (link)}. The Orbitrap Fusion was tuned and calibrated according to the manufacturer’s default standard recommendations, to routinely achieve a mass accuracy of 0.5 ppm or better. The chromatograph was outfitted with a Dionex IonPac AG11-HC-4 µm RFIC&HPIC guard (2 × 50 mm) guard column upstream of a Dionex IonPac AS11-HC-4 µm RFIC&HPIC (2 × 250 mm) column. An m/z range of 80–700 except for the organ extracts where the m/z range was 50-750. Peak areas were integrated and exported to Excel via the TraceFinder 3.3 (Themo) software package. Peak areas were corrected for natural abundance distribution of each of the isotopologues^{41 (link)}, after which fractional enrichment and µmoles metabolites/g protein were calculated to quantify ¹³C incorporation into various pathways.