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.
|Number of pages||9|
|State||Published - Feb 9 2021|
Bibliographical noteFunding Information:
This work was supported by NCI P01CA163223-01A1, 1U24DK097215-01A1, 1R01CA118434-01A2, 5R21ES025669-02, 5P20GM121327, 1P30ES026529-01A1, and Shared Resource(s) of the University of Kentucky Markey Cancer Center P30CA177558. The authors thank Dr. Ramon Sun for mouse liver reference sample preparation, Dr. Salim EI-Amouri for the A549 spheroid tracer experiment and polar extraction, Ms. Yan Zhang for the BEAS-2B cells tracer experiment and polar extraction, Dr. Jessica Macedo for recording MS data on some samples, and Dr. Marc O. Warmoes for early exploration of IC-MS. The authors also thank Mr. P. Travis Thompson for developing the TraceFinder curation method and R scripts for quantification and normalization, and Mr. Patrick Shepherd for the automated multi-isotope natural abundance correction algorithm. 2
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ASJC Scopus subject areas
- Analytical Chemistry