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

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.

The influence of redox modulation on hypoxic endothelial cell metabolic and proteomic profiles through a small thiol‐based compound tuning glutathione and thioredoxin systems

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“…Insoluble material was pelleted by centrifugation at 20,000 g for 20 min. In order to maintain redox‐sensitive metabolites, buffers for extraction followed previously published protocols 36 …”

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Folate depletion induces erythroid differentiation through perturbation of de novo purine synthesis

“…operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation and MS data acquisition was performed as previously described 42 .…”