Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 13 April 2021 / Approved: 14 April 2021 / Online: 14 April 2021 (16:09:16 CEST)

A peer-reviewed article of this Preprint also exists.

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. 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.

Journal reference: Metabolites 2021, 11, 313
DOI: 10.3390/metabo11050313

Abstract

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.

Subject Areas

redox metabolites; mass-spectrometry method; HILIC chromatography; NADH; NADPH; Glutathione; redox metabolite detection in mammalian cells

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