Ion Chromatography-MS for Metabolite Analysis

Ion chromatography- Fourier transform-MS was performed as previously described [33 (link)]. Briefly, polar extracts were reconstituted in 20 μL nanopure water, and analyzed by 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 all ¹³C isotopologues. The chromatography was performed using a Dionex IonPac AG11-HC-4 μm RFIC&HPIC (2 × 50 mm) guard column upstream of a Dionex IonPac AS11-HC-4 μm RFIC&HPIC (2 × 250 mm) column. Chromatography and mass spectrometric settings were the same as described previously [31 ] with an acquisition m/z range of 80 to 700. Metabolites and their isotopologues were identified by chromatographic retention times and their m/z values compared with those of the standards. Peak were integrated and the areas exported to Excel via the TraceFinder 3.3 (Thermo, Waltham, MA, USA) software package. Peak areas were corrected for natural abundance as previously described [34 ], after which fractional enrichment and μmoles metabolites/g protein were calculated to quantify ¹³C incorporation into various metabolites.