Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS
1
Department of Pathology, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
2
Harvard Medical School, Boston, MA 02115, USA
3
Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA
4
Broad Institute of Harvard and Massachusetts Institute of Technology, 415 Main Street, Cambridge, MA 02142, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Academic Editor: Thusitha W. Rupasinghe
Metabolites 2021, 11(5), 313; https://doi.org/10.3390/metabo11050313
Received: 13 April 2021
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Revised: 29 April 2021
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Accepted: 10 May 2021
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Published: 13 May 2021
(This article belongs to the Section Metabolomic Profiling Technology)
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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Keywords:
redox metabolites; mass-spectrometry method; HILIC chromatography; NADH; NADPH; glutathione; redox metabolite detection in mammalian cells
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MDPI and ACS Style
Petrova, B.; Warren, A.; Vital, N.Y.; Culhane, A.J.; Maynard, A.G.; Wong, A.; Kanarek, N. Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS. Metabolites 2021, 11, 313. https://doi.org/10.3390/metabo11050313
AMA Style
Petrova B, Warren A, Vital NY, Culhane AJ, Maynard AG, Wong A, Kanarek N. Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS. Metabolites. 2021; 11(5):313. https://doi.org/10.3390/metabo11050313
Chicago/Turabian StylePetrova, Boryana, Anna Warren, Nuria Y. Vital, Andrew J. Culhane, Adam G. Maynard, Alan Wong, and Naama Kanarek. 2021. "Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS" Metabolites 11, no. 5: 313. https://doi.org/10.3390/metabo11050313
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