Redox Proteomics Research Articles

Stochiometric quantification of the thiol redox proteome of macrophages reveals subcellular compartmentalization and susceptibility to oxidative perturbations

Posttranslational modifications of protein cysteine thiols play a significant role in redox regulation and the pathogenesis of human diseases. Herein, we report the characterization of the cellular redox landscape in terms of quantitative, site-specific occupancies of both S-glutathionylation (SSG) and total reversible thiol oxidation (total oxidation) in RAW 264.7 macrophage cells under basal conditions. The occupancies of thiol modifications for ~4000 cysteine sites were quantified, revealing a mean site occupancy of 4.0% for SSG and 11.9% for total oxidation, respectively. Correlations between site occupancies and structural features such as pKa, relative residue surface accessibility, and hydrophobicity were observed. Proteome-wide site occupancy analysis revealed that the average occupancies of SSG and total oxidation in specific cellular compartments correlate well with the expected redox potentials of respective organelles in macrophages, consistent with the notion of redox compartmentalization. The lowest average occupancies were observed in more reducing organelles such as the mitochondria (non-membrane) and nucleus, while the highest average occupancies were found in more oxidizing organelles such as endoplasmic reticulum (ER) and lysosome. Furthermore, a pattern of subcellular susceptibility to redox changes was observed under oxidative stress induced by exposure to engineered metal oxide nanoparticles. Peroxisome, ER, and mitochondria (membrane) are the organelles which exhibit the most significant redox changes; while mitochondria (non-membrane) and Golgi were observed as the organelles being most resistant to oxidative stress. Finally, it was observed that Cys residues at enzymatic active sites generally had a higher level of occupancy compared to non-active Cys residues within the same proteins, suggesting site occupancy as a potential indicator of protein functional sites. The raw data are available via ProteomeXchange with identifier PXD019913.

A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes.

Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining H2O2-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

P1143REDOX PROFILING IN MESOTHELIAL CELLS IN EXPERIMENTAL PERITONEAL DIALYSIS

Abstract Background and Aims PD-fluids lead to generation of reactive oxygen species (ROS) in the peritoneal cavity. The caused oxidative stress, defined as a cellular oxidant-antioxidant imbalance impairs not only peritoneal cell viability but also contributes to progression of local and systemic PD-related pathomechanisms. We aim to analyze the impact and specific targets of ROS during PD and the anti-oxidative mechanism of supplementation of PD-fluid with alanyl-glutamine (AlaGln) on a global proteome-wide level. Method To establish a redox-proteomics workflow for studying oxidative stress in peritoneal mesothelial cells we used a gold-standard model of redox-stress (H2O2) and PD-fluid induced stress. Levels of oxidative stress were first validated by increased intracellular ROS and superoxide dismutase activity with PD-fluid and H2O2 treatment and a reduction of these parameters by the addition of AlaGln. To detect alterations of the redox proteome, cysteine residues were either directly or indirectly labeled with fluorescent dyes (redox-2D-DiGE) or isobaric tags (iodo-TMT). Results: The gel-based approach allowed global visualization of the reduced and oxidized cysteines and revealed redox profiles of 540 protein spots. Compared to control, we found an increase in oxidized and decrease in reduced cysteines in all PD treatments. The development of a highly sensitive LC/MS-based redox proteomics workflow allowed identification of ∼950 proteins affected by redox-stress in mesothelial cells and confirmed the quantitative levels seen on cysteine oxidation. The addition of AlaGln reduced the overall redox status (intracellular ROS and superoxide dismutase activity) but further showed different proteins to be affected by redox modifications. Conclusion: Redox proteomics of peritoneal cells could represent a novel approach for the identification of mediators of PD-induced pathomechanisms, but also to evaluate effects of novel anti-oxidant therapeutical or pharmacological interventions.

Multi-parameter evaluation of the effect of processing conditions on meat protein modification

Evaluating the interconnecting effects of pH, temperature and time on food proteins is of relevance to food processing, and food functionality. Here we describe a matrix-based approach in which meat proteins were exposed to combinations of these parameters, selected to cover coordinates in a realistic processing space, and analyzed using redox proteomics. Regions within the matrix showing high levels of protein modification were evaluated for oxidative and other modifications. Both pH and temperature, independently, had a significant effect on the oxidative modifications mostly detected in myofibrillar proteins such as myosin and troponin and also collagen. Heat induced pyroglutamic acid formation was exclusively observed in the myofibrillar proteins. Potential interdependencies between pH, temperature and exposure time were evaluated using a 3-way analysis of variance (ANOVA) on protein modification levels to better understand how industry relevant process parameters influence protein quality and function.

Synthesis of Peptide Cysteine Dimedone Using Fmoc‐Cys(Dmd)‐OH: Glutathione Cysteine Dimedone as a Probe in Investigating the Sulfenic Acid Mediated Oxidation of Glutathione

AbstractDimedone is the most widely used chemical probe for detection of cysteine sulfenic acid in peptides and proteins. The reaction of dimedone with cysteine sulfenic acid results in the formation of unique cysteine dimedone motif containing thioether bridge. Based on the structure of cysteine dimedone residue in polypeptide, a new building block of Fmoc‐Cys(Dmd)‐OH was developed for solid phase synthesis of peptide cysteine dimedone. Mass spectrometric sequencing of synthetic peptides have confirmed successful incorporation of cysteine dimedone in peptide chain using HBTU/HOBt as a coupling agent. The new method permits synthesis of peptides containing both cysteine thiol and cysteine dimedone in the same sequence which was difficult to achieve by conventional methods. The synthetic peptide of glutathione cysteine dimedone was used as a standard in probing the air‐mediated oxidation of thiol to disulfide form of glutathione. The co‐elution of standard peptide and reaction mixture of oxidation of glutathione in presence of dimedone using RP‐HPLC have confirmed the formation of glutathione cysteine sulfenic as an intermediate in the air‐mediated oxidation of glutathione. The synthetic peptides of cysteine dimedone may find application in the field of redox proteomics and generation of antibodies against modified cysteine residue.

Metabolic Reprogramming of Mouse Bone Marrow Derived Macrophages Following Erythrophagocytosis.

Reticuloendothelial macrophages engulf ∼0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of mouse bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-13C6 glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-β, respectively. Significant increases in early Krebs cycle and C5-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.

Protein extraction from Saccharomyces cerevisiae at different growth phases

Saccharomyces cerevisiae is an established model organism with a well characterized genome. However, this model presents a unique problem due to a very resistant cell wall which develops in the late stationary phase resulting in sub-optimal extraction of proteins from such cells using majority of the cell lysis protocols. In this study, several methods from the literature with modifications thereof for lysis of S. cerevisiae cells were analyzed for their suitability for redox proteomics and biological activity studies of both exponential and late stationary phase cultures. The protocols applied are glass bead lysis, sonication, their combinations, alkali extraction, hot-SDS extraction methods and their modifications. The glass bead lysis method showed low yield but could be convenient in cases where in vitro processing steps post extraction is required or if only hydrophilic proteins are of interest. Hot-SDS and alkali extraction protocols yielded higher amount of proteins and these methods are potentially suitable for Western blotting and redox proteomic studies but allow no post-processing treatment(s) on the extracts which may be required for aging- and oxidative stress-related or other studies.

The Proteomic Landscape of Cysteine Oxidation That Underpins Retinoic Acid-Induced Neuronal Differentiation.

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.

Plant Chloroplast Stress Response: Insights from Thiol Redox Proteomics.

Significance: Plant chloroplasts generate reactive oxygen species (ROS) during photosynthesis, especially under stresses. The sulfhydryl groups of protein cysteine residues are susceptible to redox modifications, which regulate protein structure and function, and thus different signaling and metabolic processes. The ROS-governed protein thiol redox switches play important roles in chloroplasts. Recent Advances: Various high-throughput thiol redox proteomic approaches have been developed, and they have enabled the improved understanding of redox regulatory mechanisms in chloroplasts. For example, the thioredoxin-modulated antioxidant enzymes help to maintain cellular ROS homeostasis. The light- and dark-dependent redox regulation of photosynthetic electron transport, the Calvin/Benson cycle, and starch biosynthesis ensures metabolic coordination and efficient energy utilization. In addition, redox cascades link the light with the dynamic changes of metabolites in nitrate and sulfur assimilation, shikimate pathway, and biosynthesis of fatty acid hormone as well as purine, pyrimidine, and thiamine. Importantly, redox regulation of tetrapyrrole and chlorophyll biosynthesis is critical to balance the photodynamic tetrapyrrole intermediates and prevent oxidative damage. Moreover, redox regulation of diverse elongation factors, chaperones, and kinases plays an important role in the modulation of gene expression, protein conformation, and posttranslational modification that contribute to photosystem II (PSII) repair, state transition, and signaling in chloroplasts. Critical Issues: This review focuses on recent advances in plant thiol redox proteomics and redox protein networks toward understanding plant chloroplast signaling, metabolism, and stress responses. Future Directions: Using redox proteomics integrated with biochemical and molecular genetic approaches, detailed studies of cysteine residues, their redox states, cross talk with other modifications, and the functional implications will yield a holistic understanding of chloroplast stress responses.

Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura®) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.

A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging

Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology invivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome invivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

Identification of new targets of S-nitrosylation in neural stem cells by thiol redox proteomics

Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R–SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC.

Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats

Inspiratory dysfunction occurs in patients with heart failure with reduced ejection fraction (HFrEF) in a manner that depends on disease severity and by mechanisms that are not fully understood. In the current study, we tested whether HFrEF effects on diaphragm (inspiratory muscle) depend on disease severity and examined putative mechanisms for diaphragm abnormalities via global and redox proteomics. We allocated male rats into Sham, moderate (mHFrEF), or severe HFrEF (sHFrEF) induced by myocardial infarction and examined the diaphragm muscle. Both mHFrEF and sHFrEF caused atrophy in type IIa and IIb/x fibers. Maximal and twitch specific forces (N/cm2) were decreased by 19 ± 10% and 28 ± 13%, respectively, in sHFrEF (p < .05), but not in mHFrEF. Global proteomics revealed upregulation of sarcomeric proteins and downregulation of ribosomal and glucose metabolism proteins in sHFrEF. Redox proteomics showed that sHFrEF increased reversibly oxidized cysteine in cytoskeletal and thin filament proteins and methionine in skeletal muscle α-actin (range 0.5 to 3.3-fold; p < .05). In conclusion, fiber atrophy plus contractile dysfunction caused diaphragm weakness in HFrEF. Decreased ribosomal proteins and heighted reversible oxidation of protein thiols are candidate mechanisms for atrophy or anabolic resistance as well as loss of specific force in sHFrEF.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

Anticancer Effect of Deuterium Depleted Water - Redox Disbalance Leads to Oxidative Stress

Despite the convincing empirical evidence that deuterium depleted water (DDW, 25-125 ppm deuterium) has anticancer effect, the molecular mechanism remains unclear. Here, redox proteomics investigation of the DDW action in A549 cells revealed an increased level of oxidative stress, whereas expression proteomics in combination with thermal profiling uncovered crucial role of mitochondrial proteins. In the proposed scenario, reversal of the normally positive deuterium gradient across the inner membrane leads to an increased export of protons from the matrix to intermembrane space and an increase in the mitochondrial membrane potential, enhancing the production of reactive oxygen species (ROS). The resulting oxidative stress leads to slower growth and can induce apoptosis. However, further deuterium depletion in ambient water triggers a feedback mechanism, which leads to restoration of the redox equilibrium and resumed growth. The DDW-induced oxidative stress, verified by traditional biochemical assays, may be helpful as an adjuvant to ROS-inducing anticancer therapy.

The Landscape of Thiol-based Redox Proteome in terms of Occupancies of Redox Modifications and Subcellular Compartmentalization

The Landscape of Thiol-based Redox Proteome in terms of Occupancies of Redox Modifications and Subcellular Compartmentalization

Redox Proteomes in Human Physiology and Disease Mechanisms.

Redox proteomics is a field of proteomics that is concerned with the characterization of the oxidation state of proteins to gain information about their modulated structure, function, activity, and involvement in different physiological pathways. Oxidative modifications of proteins have been shown to be implicated in normal physiological processes of cells as well as in pathomechanisms leading to the development of cancer, diabetes, neurodegenerative diseases, and some rare hereditary metabolic diseases, like classic galactosemia. Reactive oxygen species generate a variety of reversible and irreversible modifications in amino acid residue side chains and within the protein backbone. These oxidative post-translational modifications (Ox-PTMs) can participate in the activation of signal transduction pathways and mediate the toxicity of harmful oxidants. Thus the application of advanced redox proteomics technologies is important for gaining insights into molecular mechanisms of diseases. Mass-spectrometry-based proteomics is one of the most powerful methods that can be used to give detailed qualitative and quantitative information on protein modifications and allows us to characterize redox proteomes associated with diseases. This Review illustrates the role and biological consequences of Ox-PTMs under basal and oxidative stress conditions by focusing on protein carbonylation and S-glutathionylation, two abundant modifications with an impact on cellular pathways that have been intensively studied during the past decade.

Redox Systems Biology: Harnessing the Sentinels of the Cysteine Redoxome.

Significance: Cellular redox processes are highly interconnected, yet not in equilibrium, and governed by a wide range of biochemical parameters. Technological advances continue refining how specific redox processes are regulated, but broad understanding of the dynamic interconnectivity between cellular redox modules remains limited. Systems biology investigates multiple components in complex environments and can provide integrative insights into the multifaceted cellular redox state. This review describes the state of the art in redox systems biology as well as provides an updated perspective and practical guide for harnessing thousands of cysteine sensors in the redoxome for multiparameter characterization of cellular redox networks. Recent Advances: Redox systems biology has been applied to genome-scale models and large public datasets, challenged common conceptions, and provided new insights that complement reductionist approaches. Advances in public knowledge and user-friendly tools for proteome-wide annotation of cysteine sentinels can now leverage cysteine redox proteomics datasets to provide spatial, functional, and protein structural information. Critical Issues: Careful consideration of available analytical approaches is needed to broadly characterize the systems-level properties of redox signaling networks and be experimentally feasible. The cysteine redoxome is an informative focal point since it integrates many aspects of redox biology. The mechanisms and redox modules governing cysteine redox regulation, cysteine oxidation assays, proteome-wide annotation of the biophysical and biochemical properties of individual cysteines, and their clinical application are discussed. Future Directions: Investigating the cysteine redoxome at a systems level will uncover new insights into the mechanisms of selectivity and context dependence of redox signaling networks.

Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H2O2-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.