Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labelling in Metabolomics
2020
Isotopic labelling experiments have been utterly
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 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.
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