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.
Highlights
Cellular redox could be defined as the highly dynamic and tightly regulated balance between molecules that function as oxidants and antioxidants
To address the need for quantification of redox metabolites in parallel to detection of polar metabolites from mammalian cells we set to optimize an Liquid-chromatography mass-spectrometry (LC-MS) method based on hydrophilic interaction chromatography (HILIC)
We focused our efforts on GSH, GSSG, NADH, nicotinamide adenine dinucleotide (NAD)+, NADPH, and nicotinamide adenine dinucleotide phosphate (NADP)+, because these metabolites are critical for redox balance and are most commonly assessed as a readout for the cellular redox state
Summary
Cellular redox could be defined as the highly dynamic and tightly regulated balance between molecules that function as oxidants and antioxidants. Cells constantly produce reactive oxygen species (ROS) through partial reduction of O2 by the electron transport chain (ETC). ROS are inactivated by enzymes such as catalase, and endogenous metabolites that function as antioxidants, such as glutathione. ROS have a role in signal transduction [1] and are important for normal physiology [2]. If ROS levels exceed their tightly-controlled normal levels, they induce oxidative stress, and can cause damage to proteins, lipids, and DNA [3]. Redox state must be finely tuned to match the specific metabolic needs of the cell or tissue
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