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-MS 1 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-MS 1 with its long acquisition time, while acquiring MS 2 data on many coeluting labeled isotopologues for each metabolite. We have developed an ion chromatography (IC)-UHR-MS 1 /data-independent(DI)-HR-MS 2 method to trace the fate of 13 C atoms from [ 13 C 6 ]-glucose ([ 13 C 6 ]-Glc) in 3D A549 spheroids in response to anticancer selenite and simultaneously 13 C/ 15 N atoms from [ 13 C 5 , 15 N 2 ]-glutamine ([ 13 C 5 , 15 N 2 ]-Gln) in 2D BEAS-2B cells in response to arsenite transformation. This method retains the complete isotopologue distributions of metabolites via UHR-MS 1 while simultaneously acquiring substructure label information via DI-MS 2 . 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.

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

“…We have applied a previously developed Ion chromatography-ultrahigh resolution Fourier transform MS 1 /DI-MS 2 method ( 21 ) for extensive and robust reconstruction of [ 13 C 6 ]-Glc or [ 13 C 5 , 15 N 2 ]-Gln–fueled central metabolic networks in mammalian cells. This method met the needs for resolving dual tracer distribution in intact metabolites with ultra high-resolution MS 1 while simultaneously acquiring positional labeling in metabolite moieties via DI-MS 2 .…”

“…All materials including the make-up solvent methanol for Ion chromatography, individual standards of metabolites used for quantification were obtained as described previously ( 21 ).…”

“…A mixture of 86 (Mix 1) and 81 (Mix 2) standards were prepared as two separate calibration standard mixtures as described previously ( 21 ). The standard mixtures were aliquoted, lyophilized, and stored at −80 °C for long term use.…”

“…Conditions for chromatographic separations ( i.e. , KOH gradient) and ion suppressor and desolvation in the heated electrospray were as described previously ( 21 ). MS data were acquired using the Xcalibur software.…”

“…This limitation prompted us to develop an Ion Chromatography-Ultrahigh Resolution-MS 1 /data independent-MS 2 (IC-UHR-MS 1 /DI-MS 2 ) method to enable determination of tracer atom position(s) in metabolite moiety by MS with higher resolution and sensitivity than NMR. This in turn allows robust reconstruction of metabolic network responses to stressors at specific enzyme levels ( 21 ). The UHR-MS 1 step is capable of resolving the neutron mass difference among different tracer atoms ( e.g.…”