Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics

Abstract

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC’s superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)­TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.

BRAF activation by metabolic stress promotes glycolysis sensitizing NRASQ61-mutated melanomas to targeted therapy

“…The samples were analyzed as previously described 55 . In brief, for isotope-labeling experiments, glucose was replaced with U- 13 C6-glucose (Sigma Aldrich).…”

Depletion of pyruvate kinase (PK) activity causes glycolytic intermediate imbalances and reveals a PK-TXNIP regulatory axis

“…Raw data was transformed into mzML open format in order to process it with isoSCAN R package [ 58 ]. Using isoSCAN the following were performed: targeted isotopologue quantification, C13 natural abundance subtraction, fractional contribution (FrC) calculation and normalization using d3-Leucine.…”

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage

“…Capellades and colleagues (2021) explored different CI reagent gases (methane and isobutane) and compared them to EI for metabolomics by GC-HRMS . The use of isobutane was discovered to prompt the [M + H] + isotopic envelope, facilitating the detection of isotopic enrichment in contrast to methane which induced unwanted [M + H] + fragmentation; no significant decline in sensitivity was observed for CI-isobutane . Meanwhile, the authors determined that a low-energy EI (15 eV) still promoted greater fragmentation of M +• than CI-isobutane .…”

“… 170 The use of isobutane was discovered to prompt the [M + H] + isotopic envelope, facilitating the detection of isotopic enrichment in contrast to methane which induced unwanted [M + H] + fragmentation; no significant decline in sensitivity was observed for CI-isobutane. 170 Meanwhile, the authors determined that a low-energy EI (15 eV) still promoted greater fragmentation of M +• than CI-isobutane. 170 Misra and Olivier (2020) compared EI-MS and CI-MS 2 (methane as reagent gas) in GC-Orbitrap HRMS.…”