Ultrahigh resolution MS¹/MS²-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS¹/data independent-MS² method to track the simultaneous incorporation of the heavy isotopes ¹³C and ¹⁵N into the moieties of purine/pyrimidine nucleotides in mammalian cells. Ultrahigh resolution-MS¹ resolves and counts multiple tracer atoms in intact metabolites, while data independent-tandem MS (MS²) determines isotopic enrichment in their moieties without concern for the numerous mass isotopologue source ions to be fragmented. Together, they enabled rigorous MS-based reconstruction of metabolic networks at specific enzyme levels. We have expanded this approach to trace the labeled atom fate of [¹³C₆]-glucose in 3D A549 spheroids in response to the anticancer agent selenite and that of [¹³C₅,¹⁵N₂]-glutamine in 2D BEAS-2B cells in response to arsenite transformation. We deduced altered activities of specific enzymes in the Krebs cycle, pentose phosphate pathway, gluconeogenesis, and UDP-GlcNAc synthesis pathways elicited by the stressors. These metabolic details help elucidate the resistance mechanism of 3D versus 2D A549 cultures to selenite and metabolic reprogramming that can mediate the transformation of BEAS-2B cells by arsenite.