Application of Stable Isotope-Assisted Metabolomics for Cell Metabolism Studies

Abstract

The applications of stable isotopes in metabolomics have facilitated the study of cell metabolisms. Stable isotope-assisted metabolomics requires: (1) properly designed tracer experiments; (2) stringent sampling and quenching protocols to minimize isotopic alternations; (3) efficient metabolite separations; (4) high resolution mass spectrometry to resolve overlapping peaks and background noises; and (5) data analysis methods and databases to decipher isotopic clusters over a broad m/z range (mass-to-charge ratio). This paper overviews mass spectrometry based techniques for precise determination of metabolites and their isotopologues. It also discusses applications of isotopic approaches to track substrate utilization, identify unknown metabolites and their chemical formulas, measure metabolite concentrations, determine putative metabolic pathways, and investigate microbial community populations and their carbon assimilation patterns. In addition, 13C-metabolite fingerprinting and metabolic models can be integrated to quantify carbon fluxes (enzyme reaction rates). The fluxome, in combination with other “omics” analyses, may give systems-level insights into regulatory mechanisms underlying gene functions. More importantly, 13C-tracer experiments significantly improve the potential of low-resolution gas chromatography-mass spectrometry (GC-MS) for broad-scope metabolism studies. We foresee the isotope-assisted metabolomics to be an indispensable tool in industrial biotechnology, environmental microbiology, and medical research.

Metabolite profiling of sheath blight disease resistance in rice: in the case of positive ion mode analysis by CE/TOF-MS

“…Based on the result, it suggested that CE/TOF-MS study can be used as an option to replace the function of stable isotope labelling as carbon flow tracer in the plant. However, Kluger et al (2014) and You et al (2014) explained that stable isotope labelling is more effective to trace the metabolite element in cells.…”

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“…7 Isotope-assisted metabolomics is applicable for certain classes of metabolites or simplified metabolite mixtures that allow measurement and interpretation of endogenous and released metabolites flux in cells. 27 The MRM analysis of glycolytic pathway and TCA intermediate metabolites, such as G6P, a-KG, and malate, demonstrated a significant decrease in HIV-1 Vpr transduced macrophages compared to control cells. This suggests an increase in the enzymatic activity of the respective enzyme that consumes the intermediate metabolite and is in agreement with our earlier observation that HIV-1 Vpr induced the protein levels of the glycolytic pathway and TCA cycle.…”

Deuterium‐ and Tritium‐Labelled Compounds: Applications in the Life Sciences

“…[207] In this context, metabolic isotope fingerprinting or footprinting are frequently used terminologies in metabolomics analysis to distinguish intracellular and extracellular metabolite profiling. [208] Thed etermination of metabolic fluxes provides detailed information on cellular physiology. [209] Furthermore,t he assessment of metabolic flux changes upon ac ertain perturbation can provide novel information relating to the regulation of pathways [210] as well as their roles in growth, [211] toxicology, [212] and disease, [213] and thus directly facilitates the improvement of biotechnological processes [214] and clinical research.…”