An Ion Chromatography–Ultrahigh-Resolution-MS1/Data-Independent High-Resolution MS2 Method for Stable Isotope-Resolved Metabolomics Reconstruction of Central Metabolic Networks

Abstract

The metabolome comprises a complex network of interconnecting enzyme-catalyzed reactions that involve transfers of numerous molecular subunits. Thus, the reconstruction of metabolic networks requires metabolite substructures to be tracked. Subunit tracking can be achieved by tracing stable isotopes through metabolic transformations using NMR and ultrahigh -resolution (UHR)-mass spectrometry (MS). UHR-MS¹ readily resolves and counts isotopic labels in metabolites but requires tandem MS to help identify isotopic enrichment in substructures. However, it is challenging to perform chromatography-based UHR-MS¹ with its long acquisition time, while acquiring MS² data on many coeluting labeled isotopologues for each metabolite. We have developed an ion chromatography (IC)-UHR-MS¹/data-independent(DI)-HR-MS² method to trace the fate of ¹³C atoms from [¹³C₆]-glucose ([¹³C₆]-Glc) in 3D A549 spheroids in response to anticancer selenite and simultaneously ¹³C/¹⁵N atoms from [¹³C₅,¹⁵N₂]-glutamine ([¹³C₅,¹⁵N₂]-Gln) in 2D BEAS-2B cells in response to arsenite transformation. This method retains the complete isotopologue distributions of metabolites via UHR-MS¹ while simultaneously acquiring substructure label information via DI-MS². These details in metabolite labeling patterns greatly facilitate rigorous reconstruction of multiple, intersecting metabolic pathways of central metabolism, which are illustrated here for the purine/pyrimidine nucleotide biosynthesis. The pathways reconstructed based on subunit-level isotopologue analysis further reveal specific enzyme-catalyzed reactions that are impacted by selenite or arsenite treatments.