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Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring

[Image: see text] Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex...

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Autores principales: Huang, Luojiao, Drouin, Nicolas, Causon, Jason, Wegrzyn, Agnieszka, Castro-Perez, Jose, Fleming, Ronan, Harms, Amy, Hankemeier, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933045/
https://www.ncbi.nlm.nih.gov/pubmed/36735349
http://dx.doi.org/10.1021/acs.analchem.2c04231
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author Huang, Luojiao
Drouin, Nicolas
Causon, Jason
Wegrzyn, Agnieszka
Castro-Perez, Jose
Fleming, Ronan
Harms, Amy
Hankemeier, Thomas
author_facet Huang, Luojiao
Drouin, Nicolas
Causon, Jason
Wegrzyn, Agnieszka
Castro-Perez, Jose
Fleming, Ronan
Harms, Amy
Hankemeier, Thomas
author_sort Huang, Luojiao
collection PubMed
description [Image: see text] Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS(2) fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRM(HR)). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS(2) fragmentation mode based on the measurement of (13)C- or (15)N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of (13)C/(15)N atoms from D-(13)C(6)-glucose/L-(15)N(2)-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration.
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spelling pubmed-99330452023-02-17 Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring Huang, Luojiao Drouin, Nicolas Causon, Jason Wegrzyn, Agnieszka Castro-Perez, Jose Fleming, Ronan Harms, Amy Hankemeier, Thomas Anal Chem [Image: see text] Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS(2) fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRM(HR)). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS(2) fragmentation mode based on the measurement of (13)C- or (15)N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of (13)C/(15)N atoms from D-(13)C(6)-glucose/L-(15)N(2)-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration. American Chemical Society 2023-02-03 /pmc/articles/PMC9933045/ /pubmed/36735349 http://dx.doi.org/10.1021/acs.analchem.2c04231 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Huang, Luojiao
Drouin, Nicolas
Causon, Jason
Wegrzyn, Agnieszka
Castro-Perez, Jose
Fleming, Ronan
Harms, Amy
Hankemeier, Thomas
Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring
title Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring
title_full Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring
title_fullStr Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring
title_full_unstemmed Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring
title_short Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring
title_sort reconstruction of glutathione metabolism in the neuronal model of rotenone-induced neurodegeneration using mass isotopologue analysis with hydrophilic interaction liquid chromatography-zeno high-resolution multiple reaction monitoring
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933045/
https://www.ncbi.nlm.nih.gov/pubmed/36735349
http://dx.doi.org/10.1021/acs.analchem.2c04231
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