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Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry

[Image: see text] (13)C-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 comp...

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Autores principales: Evers, Bernard, Gerding, Albert, Boer, Theo, Heiner-Fokkema, M. Rebecca, Jalving, Mathilde, Wahl, S. Aljoscha, Reijngoud, Dirk-Jan, Bakker, Barbara M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253487/
https://www.ncbi.nlm.nih.gov/pubmed/34060804
http://dx.doi.org/10.1021/acs.analchem.1c01040
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author Evers, Bernard
Gerding, Albert
Boer, Theo
Heiner-Fokkema, M. Rebecca
Jalving, Mathilde
Wahl, S. Aljoscha
Reijngoud, Dirk-Jan
Bakker, Barbara M.
author_facet Evers, Bernard
Gerding, Albert
Boer, Theo
Heiner-Fokkema, M. Rebecca
Jalving, Mathilde
Wahl, S. Aljoscha
Reijngoud, Dirk-Jan
Bakker, Barbara M.
author_sort Evers, Bernard
collection PubMed
description [Image: see text] (13)C-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 (13)C-isotopically labeled sample is required to quantify (13)C-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 (13)C-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 (13)C-isotope enrichments in a single (13)C-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 (13)C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and (13)C-isotope enrichments in a single (13)C-isotopically labeled sample.
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spelling pubmed-82534872021-07-06 Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry Evers, Bernard Gerding, Albert Boer, Theo Heiner-Fokkema, M. Rebecca Jalving, Mathilde Wahl, S. Aljoscha Reijngoud, Dirk-Jan Bakker, Barbara M. Anal Chem [Image: see text] (13)C-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 (13)C-isotopically labeled sample is required to quantify (13)C-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 (13)C-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 (13)C-isotope enrichments in a single (13)C-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 (13)C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and (13)C-isotope enrichments in a single (13)C-isotopically labeled sample. American Chemical Society 2021-06-01 2021-06-15 /pmc/articles/PMC8253487/ /pubmed/34060804 http://dx.doi.org/10.1021/acs.analchem.1c01040 Text en © 2021 The Authors. Published by American Chemical Society Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Evers, Bernard
Gerding, Albert
Boer, Theo
Heiner-Fokkema, M. Rebecca
Jalving, Mathilde
Wahl, S. Aljoscha
Reijngoud, Dirk-Jan
Bakker, Barbara M.
Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry
title Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry
title_full Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry
title_fullStr Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry
title_full_unstemmed Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry
title_short Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry
title_sort simultaneous quantification of the concentration and carbon isotopologue distribution of polar metabolites in a single analysis by gas chromatography and mass spectrometry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253487/
https://www.ncbi.nlm.nih.gov/pubmed/34060804
http://dx.doi.org/10.1021/acs.analchem.1c01040
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