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

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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.

Original languageEnglish
Article number102586
JournalJournal of Biological Chemistry
Issue number12
StatePublished - Dec 2022

Bibliographical note

Funding Information:
This work was supported by NCI P01CA163223-01A1 , 1U24DK097215-01A1 , 1R01CA118434-01A2 , 5R21ES025669-02 , 5P20GM121327 , 5P30ES026529 , and Shared Resource(s) of the University of Kentucky Markey Cancer Center P30CA177558 . We thank Dr Salim EI-Amouri for assistance in the A549 spheroid tracer experiment and polar extraction and Ms Yan Zhang for the BEAS-2B cells tracer experiment and polar extraction. We also thank Dr Marc O. Warmoes, Patrick Shepherd, and Travis Thompson for developing the TraceFinder curation method and R scripts for automatic natural abundance correction, quantification, and normalization and Dr A. Lane for comments on the article.

Publisher Copyright:
© 2022 The Authors


  • [C,N]-glutamine
  • [C]-glucose
  • arsenite
  • metabolic pathway reconstruction
  • positional isotopologues
  • selenite
  • stable isotope resolved metabolomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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