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Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react w...

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Autores principales: Petrova, Boryana, Warren, Anna, Vital, Nuria Yulia, Culhane, Andrew J., Maynard, Adam G., Wong, Alan, Kanarek, Naama
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153172/
https://www.ncbi.nlm.nih.gov/pubmed/34068241
http://dx.doi.org/10.3390/metabo11050313
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author Petrova, Boryana
Warren, Anna
Vital, Nuria Yulia
Culhane, Andrew J.
Maynard, Adam G.
Wong, Alan
Kanarek, Naama
author_facet Petrova, Boryana
Warren, Anna
Vital, Nuria Yulia
Culhane, Andrew J.
Maynard, Adam G.
Wong, Alan
Kanarek, Naama
author_sort Petrova, Boryana
collection PubMed
description Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.
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spelling pubmed-81531722021-05-27 Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS Petrova, Boryana Warren, Anna Vital, Nuria Yulia Culhane, Andrew J. Maynard, Adam G. Wong, Alan Kanarek, Naama Metabolites Article Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell’s metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields. MDPI 2021-05-13 /pmc/articles/PMC8153172/ /pubmed/34068241 http://dx.doi.org/10.3390/metabo11050313 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Petrova, Boryana
Warren, Anna
Vital, Nuria Yulia
Culhane, Andrew J.
Maynard, Adam G.
Wong, Alan
Kanarek, Naama
Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
title Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
title_full Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
title_fullStr Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
title_full_unstemmed Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
title_short Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
title_sort redox metabolism measurement in mammalian cells and tissues by lc-ms
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153172/
https://www.ncbi.nlm.nih.gov/pubmed/34068241
http://dx.doi.org/10.3390/metabo11050313
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