13C-isotope tracing is a frequently employed approach to study metabolic pathway activity. When combined with the subsequent quantification of absolute metabolite concentrations, this enables detailed characterization of the metabolome in biological specimens and facilitates computational time-resolved flux quantification. Classically, a 13C-isotopically labeled sample is required to quantify 13C-isotope enrichments and a second unlabeled sample for the quantification of metabolite concentrations. The rationale for a second unlabeled sample is that the current methods for metabolite quantification rely mostly on isotope dilution mass spectrometry (IDMS) and thus isotopically labeled internal standards are added to the unlabeled sample. This excludes the absolute quantification of metabolite concentrations in 13C-isotopically labeled samples. To address this issue, we have developed and validated a new strategy using an unlabeled internal standard to simultaneously quantify metabolite concentrations and 13C-isotope enrichments in a single 13C-labeled sample based on gas chromatography−mass spectrometry (GC/MS). The method was optimized for amino acids and citric acid cycle intermediates and was shown to have high analytical precision and accuracy. Metabolite concentrations could be quantified in small tissue samples (≥20 mg). Also, we applied the method on 13C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and 13C-isotope enrichments in a single 13C-isotopically labeled sample.
Bibliographical noteFunding Information:
This work was funded by the UMCG. Furthermore, this study was supported by a Dutch Cancer Society grant awarded to Mathilde Jalving (KWF 10913/2017-1) and a grant from the European Union Horizon 2020 Research and Innovation Program to Barbara Bakker (MESI-STRAT project, grant agreement 754688).
© 2021 The Authors. Published by American Chemical Society
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