Oxidative stress is biochemically complex. Like primary colours, specific reactive oxygen species (ROS) and antioxidant inputs can be mixed to create unique "shades" of oxidative stress. Even a minimal redox module comprised of just 12 (ROS & antioxidant) inputs and 3 outputs (oxidative damage, cysteine-dependent redox-regulation, or both) yields over half a million "shades" of oxidative stress. The present paper proposes the novel hypothesis that: state-specific shades of oxidative stress, such as a discrete disease, are associated with distinct tell-tale cysteine oxidation patterns. The patterns are encoded by many parameters, from the identity of the oxidised proteins, the cysteine oxidation type, and magnitude. The hypothesis is conceptually grounded in distinct ROS and antioxidant inputs coalescing to produce unique cysteine oxidation outputs. And considers the potential biological significance of the holistic cysteine oxidation outputs. The literature supports the existence of state-specific cysteine oxidation patterns. Measuring and manipulating these patterns offer promising avenues for advancing oxidative stress research. The pattern inspired hypothesis provides a framework for understanding the complex biochemical nature of state-specific oxidative stress.

Environmental proteases have been widely associated to the pathogenesis of allergic disorders. Der p 1, a cysteine-protease from house dust mite (HDM) Dermatophagoides pteronyssinus, constitutes one of the most clinically relevant indoor aeroallergens worldwide. Der p 1 protease activity depends on the redox status of its catalytic cysteine residue, which has to be in the reduced state to be active. So far, it is unknown whether Der p 1-protease activity could be regulated by host redox microenvironment once it reaches the lung epithelial lining fluid in addition to endogenous mite components. In this sense, Glutathione-S-transferase pi (GSTpi), an enzyme traditionally linked to phase II detoxification, is highly expressed in human lung epithelial cells, which represent the first line of defence against aeroallergens. Moreover, GSTpi is a generalist catalyst of protein S-glutathionylation reactions, and some polymorphic variants of this enzyme has been associated to the development of allergic asthma. Here, we showed that human GSTpi increased the cysteine-protease activity of Der p 1, while GSTmu (the isoenzyme produced by the mite) did not alter it. GSTpi induces the reduction of Cys residues in Der p 1, probably by rearranging its disulphide bridges. Furthermore, GSTpi was detected in the apical medium collected from human bronchial epithelial cell cultures, and more interesting, it increased cysteine-protease activity of Der p 1. Our findings support the role of human GSTpi from airways in modulating of Der p 1 cysteine-protease activity, which may have important clinical implications for immune response to this aeroallergen in genetically susceptible individuals.

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.

Innate immunity is the first line of host defense against pathogens. This process is modulated by multiple antiviral protein modifications, such as phosphorylation and ubiquitination. Here, we showed that cellular S-nitrosoglutathione reductase (GSNOR) is actively involved in innate immunity activation. GSNOR deficiency in mouse embryo fibroblasts (MEFs) and RAW264.7 macrophages reduced the antiviral innate immune response and facilitated herpes simplex virus-1 (HSV-1) and vesicular stomatitis virus (VSV) replication. Concordantly, HSV-1 infection in Gsnor-/- mice and wild-type mice with GSNOR being inhibited by N6022 resulted in higher mortality relative to the respective controls, together with severe infiltration of immune cells in the lungs. Mechanistically, GSNOR deficiency enhanced cellular TANK-binding kinase 1 (TBK1) protein S-nitrosation at the Cys423 site and inhibited TBK1 kinase activity, resulting in reduced interferon production for antiviral responses. Our study indicated that GSNOR is a critical regulator of antiviral responses and S-nitrosation is actively involved in innate immunity.

Redox active cysteine residues including βCys93 are part of hemoglobin's "oxidation hotspot". Irreversible oxidation of βCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.

The cellular effects of hydrogen sulfide (H2S) signaling may be partially mediated by the formation of alkyl persulfides from thiols, such as glutathione and protein cysteine residues. Persulfides are potent nucleophiles and reductants and therefore potentially an important endogenous antioxidant or protein post-translational modification. To directly study the cellular effects of persulfides, cysteine trisulfide (Cys-S3) has been proposed as an in situ persulfide donor, as it reacts with cellular thiols to generate cysteine persulfide (Cys-S-S-). Numerous pathways sense and respond to electrophilic cellular stressors to inhibit cellular proliferation and induce apoptosis, however the effect of Cys-S3 on the cellular stress response has not been addressed. Here we show that Cys-S3 inhibited cellular metabolism and proliferation and rapidly induced cellular- and ER-stress mechanisms, which were coupled to widespread protein-thiol oxidation. Cys-S3 reacted with Na2S to generate cysteine persulfide, which protected human cell lines from ER-stress. However this method of producing cysteine persulfide contains excess sulfide, which interferes with the direct analysis of persulfide donation. We conclude that cysteine trisulfide is a thiol oxidant that induces cellular stress and decreased proliferation.

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions.

Mitochondrial dysfunction is related to common age-related disorders, including neurodegenerative diseases, metabolic syndrome, and carcinogenesis. Therefore, maintaining the functionality and integrity of mitochondria is important for human health. Herein, we found that sulfide:quinone oxidoreductase (Sqr), which oxidizes hydrogen sulfide to reactive sulfur species (RSS), was indispensable to mitochondria health in the eukaryotic model microorganism Schizosaccharomyces pombe. Sqr knock-out led to morphological changes and functional deficiencies of mitochondria and apoptosis in S. pombe. The Sqr knock-out strain displayed the same phenotypes as the cysteine-synthesis-deficient strain, and cysteine addition complemented the effects caused by Sqr knock-out. In S. pombe, Sqr was the main RSS producer in mitochondria, and RSS instead of H2S was used by cysteine synthase to synthesize cysteine. This finding rewrites the cysteine biosynthesis route in S. pombe and may also in other eukaryotes and prokaryotes, and highlights the importance of cysteine and RSS in maintaining mitochondrial health.

Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn+2 binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism.

Preterm infants and patients with lung disease often have excess fluid in the lungs and are frequently treated with oxygen, however long-term exposure to hyperoxia results in irreversible lung injury. Although the adverse effects of hyperoxia are mediated by reactive oxygen species, the full extent of the impact of hyperoxia on redox-dependent regulation in the lung is unclear. In this study, neonatal mice overexpressing the beta-subunit of the epithelial sodium channel (β-ENaC) encoded by Scnn1b and their wild type (WT; C57Bl6) littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FiO2)-induced lung injury. Results showed that O2-induced lung injury in transgenic Scnn1b mice is attenuated following chronic O2 exposure. To test the hypothesis that reversible cysteine-redox-modifications of proteins play an important role in O2-induced lung injury, we performed proteome-wide profiling of protein S-glutathionylation (SSG) in both WT and Scnn1b overexpressing mice maintained at 21% O2 (normoxia) or FiO2 85% (hyperoxia) from birth to 11-15 days postnatal. Over 7700 unique Cys sites with SSG modifications were identified and quantified, covering more than 3000 proteins in the lung. In both mouse models, hyperoxia resulted in a significant alteration of the SSG levels of Cys sites belonging to a diverse range of proteins. In addition, substantial SSG changes were observed in the Scnn1b overexpressing mice exposed to hyperoxia, suggesting that ENaC plays a critically important role in cellular regulation. Hyperoxia-induced SSG changes were further supported by the results observed for thiol total oxidation, the overall level of reversible oxidation on protein cysteine residues. Differential analyses reveal that Scnn1b overexpression may protect against hyperoxia-induced lung injury via modulation of specific processes such as cell adhesion, blood coagulation, and proteolysis. This study provides a landscape view of protein oxidation in the lung and highlights the importance of redox regulation in O2-induced lung injury.

The reversible oxidation of protein cysteine residues (Cys-SH) is a key reaction in cellular redox signaling involving initial formation of sulfenic acids (Cys-SOH), which are commonly detected using selective dimedone-based probes. Here, we report that significant portions of dimedone-tagged proteins are susceptible to cleavage by DTT reflecting the presence of perthiosulfenic acid species (Cys-SSOH) due to similar oxidation of hydropersulfides (Cys-SSH), since Cys-S-dimedone adducts are stable toward DTT. Combined studies using molecular modeling, mass spectrometry, and cell-based experiments indicate that Cys-SSH are readily oxidized to Cys-SSOH, which forms stable adducts with dimedone-based probes. We additionally confirm the presence of Cys-SSH within protein tyrosine kinases such as EGFR, and their apparent oxidation to Cys-SSOH in response NADPH oxidase activation, suggesting that such Cys-SSH oxidation may represent a novel, as yet uncharacterized, event in redox-based signaling.

Currently the field of epilepsy lacks peripheral blood-based biomarkers that could predict the onset or progression of chronic seizures following an epileptogenic injury. Thiol/disulfide ratios have been shown to provide a sensitive means of assessing the systemic redox potential in tissue and plasma. In this study, we utilized a rapid, simple and reliable method for simultaneous determination of several thiol-containing amino acids in plasma using HPLC with electrochemical detection in kainic acid (KA) and pilocarpine rat models of epilepsy. In contrast to GSH and GSSG levels, the levels of cysteine (Cys) were decreased by 42% and 62% and cystine (Cyss) were increased by 46% and 23% in the plasma of KA- and pilocarpine-injected rats, respectively after 48h. In chronically epileptic rats, plasma cysteine was decreased by 40.4% and 37.7%, and plasma GSSG increased by 33.8% and 35.0% following KA and pilocarpine, respectively. Treatment of rats with a catalytic antioxidant, 60min after KA or pilocarpine significant attenuated the decrease of plasma Cys/Cyss ratios at the 48h time point in both models. These observations suggest that the decreased cysteine and ratio of Cys/Cyss in plasma could potentially serve as redox biomarkers in temporal lobe epilepsy.

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress, which induce extensive reorganization of the vimentin cytoskeletal network. Here, we have investigated the mechanisms underlying these effects. In vitro, electrophilic lipids, including 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal (HNE), directly bind to vimentin, whereas the oxidant diamide induces disulfide bond formation. Mutation of the single vimentin cysteine residue (Cys328) blunts disulfide formation and reduces lipoxidation by 15d-PGJ2, but not HNE. Preincubation with these agents differentially hinders NaCl-induced filament formation by wild-type vimentin, with effects ranging from delayed elongation and increased filament diameter to severe impairment of assembly or aggregation. Conversely, the morphology of vimentin Cys328Ser filaments is mildly or not affected. Interestingly, preformed vimentin filaments are more resistant to electrophile-induced disruption, although chemical modification is not diminished, showing that vimentin (lip)oxidation prior to assembly is more deleterious. In cells, electrophiles, particularly diamide, induce a fast and drastic disruption of existing filaments, which requires the presence of Cys328. As the cellular vimentin network is under continuous remodeling, we hypothesized that vimentin exchange on filaments would be necessary for diamide-induced disruption. We confirmed that strategies reducing vimentin dynamics, as monitored by FRAP, including cysteine crosslinking and ATP synthesis inhibition, prevent diamide effect. In turn, phosphorylation may promote vimentin disassembly. Indeed, treatment with the phosphatase inhibitor calyculin A to prevent dephosphorylation intensifies electrophile-induced wild-type vimentin filament disruption. However, whereas a phosphorylation-deficient vimentin mutant is only partially protected from disorganization, Cys328Ser vimentin is virtually resistant, even in the presence of calyculin A. Together, these results indicate that modification of Cys328 and vimentin exchange are critical for electrophile-induced network disruption.

Ferroptosis is inhibited by glutathione peroxidase 4 (GPX4), an antioxidant enzyme that uses reduced glutathione (GSH) as a cofactor to detoxify lipid hydroperoxides. As a selenoprotein, the core function of GPX4 is the thiol-dependent redox reaction. In addition to GSH, other small molecules such as cysteine and homocysteine also contain thiols; yet, whether GPX4 can exploit cysteine and homocysteine to directly detoxify lipid hydroperoxides and inhibit ferroptosis has not been addressed. In this study, we found that cysteine and homocysteine inhibit ferroptosis in a GPX4-dependent manner. However, cysteine inhibits ferroptosis independent of GSH synthesis, and homocysteine inhibits ferroptosis through non-cysteine and non-GSH pathway. Furthermore, we used molecular docking and GPX4 activity analysis to study the binding patterns and affinity between GPX4 and GSH, cysteine, and homocysteine. We found that besides GSH, cysteine and homocysteine are also able to serve as substrates for GPX4 though the affinities of GPX4 with cysteine and homocysteine are lower than that with GSH. Importantly, GPX family and the GSH synthetase pathway might be asynchronously evolved. When GSH synthetase is absent, for example in Flexibacter, the fGPX exhibits higher affinity with cysteine and homocysteine than GSH. Taken together, the present study provided the understanding of the role of thiol-dependent redox systems in protecting cells from ferroptosis and propose that GSH might be a substitute for cysteine or homocysteine to be used as a cofactor for GPX4 during the evolution of aerobic metabolism.

In the U.S., alcohol-associated liver disease (ALD) impacts millions of people and is a major healthcare burden. While the pathology of ALD is unmistakable, the molecular mechanisms underlying ethanol hepatotoxicity are not fully understood. Hepatic ethanol metabolism is intimately linked with alterations in extracellular and intracellular metabolic processes, specifically oxidation/reduction reactions. The xenobiotic detoxification of ethanol leads to significant disruptions in glycolysis, β-oxidation, and the TCA cycle, as well as oxidative stress. Perturbation of these regulatory networks impacts the redox status of critical regulatory protein thiols throughout the cell. Integrating these key concepts, our goal was to apply a cutting-edge approach toward understanding mechanisms of ethanol metabolism in disrupting hepatic thiol redox signaling. Utilizing a chronic murine model of ALD, we applied a cysteine targeted click chemistry enrichment coupled with quantitative nano HPLC-MS/MS to assess the thiol redox proteome. Our strategy reveals that ethanol metabolism largely reduces the cysteine proteome, with 593 cysteine residues significantly reduced and 8 significantly oxidized cysteines. Ingenuity Pathway Analysis demonstrates that ethanol metabolism reduces specific cysteines throughout ethanol metabolism (Adh1, Cat, Aldh2), antioxidant pathways (Prx1, Mgst1, Gsr), as well as many other biochemical pathways. Interestingly, a sequence motif analysis of reduced cysteines showed a correlation for hydrophilic, charged amino acids lysine or glutamic acid nearby. Further research is needed to determine how a reduced cysteine proteome impacts individual protein activity across these protein targets and pathways. Additionally, understanding how a complex array of cysteine-targeted post-translational modifications (e.g., S-NO, S-GSH, S-OH) are integrated to regulate redox signaling and control throughout the cell is key to the development of redox-centric therapeutic agents targeted to ameliorate the progression of ALD.

Oxidative modification of cysteine residues has been shown to regulate the activity of several protein-tyrosine kinases. We explored the possibility that Fms-like tyrosine kinase 3 (FLT3), a hematopoietic receptor-tyrosine kinase, is subject to this type of regulation. An underlying rationale was that the FLT3 gene is frequently mutated in Acute Myeloid Leukemia patients, and resulting oncogenic variants of FLT3 with 'internal tandem duplications (FLT3ITD)' drive production of reactive oxygen in leukemic cells. FLT3 was moderately activated by treatment of intact cells with hydrogen peroxide. Conversely, FLT3ITD signaling was attenuated by cell treatments with agents inhibiting formation of reactive oxygen species. FLT3 and FLT3ITD incorporated DCP-Bio1, a reagent specifically reacting with sulfenic acid residues. Mutation of FLT3ITD cysteines 695 and 790 reduced DCP-Bio1 incorporation, suggesting that these sites are subject to oxidative modification. Functional characterization of individual FLT3ITD cysteine-to-serine mutants of all 8 cytoplasmic cysteines revealed phenotypes in kinase activity, signal transduction and cell transformation. Replacement of cysteines 681, 694, 695, 807, 925, and 945 attenuated signaling and blocked FLT3ITD-mediated cell transformation, whereas mutation of cysteine 790 enhanced activity of both FLT3ITD and wild-type FLT3. These effects were not related to altered FLT3ITD dimerization, but likely caused by changed intramolecular interactions. The findings identify the functional relevance of all cytoplasmic FLT3ITD cysteines, and indicate the potential for redox regulation of this clinically important oncoprotein.

Herein, a novel fluorescent probe RhoDCM was developed for monitoring the cysteine (Cys) dynamics. For the first time, the Cys-triggered implement was applied in relatively complete diabetic mice models. The response of RhoDCM towards Cys suggested advantages including practical sensitivity, high selectivity, rapid reaction, and steadiness in various pH and temperature conditions. RhoDCM could basically monitor the intracellular Cys level, both exogenous and endogenous. It could further monitor the glucose level via detecting consumed Cys. Furthermore, the diabetic mice models including the no diabetic control group, the induced model groups by streptozocin (STZ) or alloxan, and the treatment groups induced by STZ and treated with vildagliptin (Vil), dapagliflozin (DA), or metformin (Metf) were constructed. The models were checked by oral glucose tolerance test and significant liver-related serum indexes. Based on the models, the in vivo imaging and penetrating depth fluorescence imaging both indicated that RhoDCM could characterize the status of the development and treatment in the diabetic process via monitoring the Cys dynamics. Consequently, RhoDCM seemed beneficial for inferring the order of severity in the diabetic process and evaluating the potency of therapeutic schedules, which might be informatic for correlated investigations.

The (seleno)cysteine residues in some protein families react with hydroperoxides with rate constants far beyond those of fully dissociated low molecular weight thiol or selenol compounds. In case of the glutathione peroxidases, we could demonstrate that high rate constants are achieved by a proton transfer from the chalcogenol to a residue of the active site [Orian et al. Free Radic. Biol. Med. 87 (2015)]. We extended this study to three more protein families (OxyR, GAPDH and Prx). According to DFT calculations, a proton transfer from the active site chalcogenol to a residue within the active site is a prerequisite for both, creating a chalcogenolate that attacks one oxygen of the hydroperoxide substrate and combining the delocalized proton with the remaining OH or OR, respectively, to create an ideal leaving group. The "parking postions" of the delocalized proton differ between the protein families. It is the ring nitrogen of tryptophan in GPx, a histidine in GAPDH and OxyR and a threonine in Prx. The basic principle, however, is common to all four families of proteins. We, thus, conclude that the principle outlined in this investigation offers a convincing explanation for how a cysteine residue can become peroxidatic.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder. Hydrogen sulfide (H2S), the third physiological gasotransmitter, is well recognized as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the protective effects of S-propargyl-cysteine (SPRC, also known as ZYZ-802), an endogenous H2S modulator, on RA and determined the underlying mechanisms. In the present study, SPRC concentration-dependently attenuated inflammatory mediator expression, reactive oxidase species generation, and the expression and activity of matrix metalloproteinases (MMP)-9 in interleukin (IL)-1β-induced human rheumatoid fibroblast-like synoviocytes MH7A. In addition, SPRC blocked IL-1β-mediated migration and invasion of MH7A cells. As expected, the protective effects of SPRC were partially abrogated by DL-propargylglycine (PAG, a H2S biosynthesis inhibitor). In vivo study also demonstrated that SPRC treatment markedly ameliorated the severity of RA in adjuvant-induced arthritis rats, and this effect was associated with the inhibition of inflammatory response. We further identified that SPRC remarkably induced heme oxygenase-1 expression associated with the degradation of Kelch-like ECH-associated protein 1 (Keap1) and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); this effect was attributed to the sulfhydrylation of the cysteine residue of Keap1. Our data demonstrated for the first time that SPRC, an endogenous H2S modulator, exerted anti-inflammatory properties in RA by upregulating the Nrf2-antioxidant response element (ARE) signaling pathway.

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.