Formation Of Cysteine Sulfenic Acid Research Articles

S-nitrosylation and S-glutathionylation of GAPDH: Similarities, differences, and relationships

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation.Incubation of HEK 293 T cells in the presence of H2O2 or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H2O2 or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.

Valproic Acid Causes an Increase in S-glutathionylation and Cysteine Sulfenic Acid Formation in vitro

Valproic Acid Causes an Increase in S-glutathionylation and Cysteine Sulfenic Acid Formation in vitro

Synthesis of two closely spaced cysteine barbiturates containing peptides by copper-catalyzed oxidation of contryphan disulfide

ABSTRACTIn this report, we are documenting the synthesis of peptide barbiturate through copper-catalyzed oxidation of peptide disulfide. Single disulfide-containing contryphans are used as models to access possibility of anchoring of barbituric acid (BRB) onto the peptide disulfide. Current method permits anchoring of two molecules of BRB onto the polypeptide and yield of peptide barbiturates varies from 59 to 84%. Formation of cysteine sulfenic acid (Cys-SOH) during oxidation of disulfide was confirmed using chemical probe of Cys-SOH dimedone. Mass spectrometric studies have confirmed the presence of cysteine barbiturate in anchored peptides. Based on the nature of reactive oxygen species involved in oxidation of peptide disulfide, possible mechanisms were proposed for anchoring of BRB onto the peptide disulfide through Cys-SOH. This is the first report on anchoring of two molecules of BRB onto the closely spaced cysteine residues of single polypeptide.

75 - VEGFR2 Signaling Driven by Cellular Redox Status

Angiogenesis (a process of new vessel formation) is triggered by tissue ischemia. Paradoxically, angiogenesis is diminished in cardiovascular diseases (CVDs) due to a decrease in cellular glutathione (GSH) and increase in reactive oxygen species (ROS). Interestingly, our laboratory has demonstrated that a small decrease in cellular GSH promotes vascular endothelial growth factor (VEGF)-driven ischemic angiogenesis while a larger decrease in cellular GSH does not. Thus, we hypothesize VEGF receptor2 (VEGFR2) activation is regulated by intracellular GSH redox state. To specifically study the role of altered GSH redox state on angiogenesis, we used diamide to oxidize GSH to GSSG in human aortic endothelial cells. GSSG can react with protein cysteine thiols to form S-glutathionylated proteins that in turn affect protein function and stability. Our preliminary experiments show that drastic changes in GSH/GSSG activate VEGFR2 independent of VEGF while a moderate change does not activate the receptor. Interestingly, the addition of H2O2 alone did not activate VEGFR2 suggesting a role for GSH/GSSG in regulating VEGFR2. To understand the mechanism of S-glutathionylation in VEGFR2 activation, we used dimedone, a sulfenic acid trap. Pretreatment with dimedone abolished VEGFR2 activation after diamide treatment suggesting that cysteine sulfenic acid formation precedes S-glutathionylation. Local ROS production in the vicinity of proteins can effectively mediate cellular signaling. On that note, O2●– derived from endothelial nitric oxide synthase (eNOS) could activate VEGFR2 in altered GSH/GSSG. S-glutathionylation of eNOS leads to its uncoupling and drives O2●– generation. To test the role of O2●– in VEGFR2 activation in altered GSH redox state, we inhibited eNOS using L-NAME or siRNA and then treated with diamide. VEGFR2 activation decreased after eNOS inhibition suggesting a potential role for eNOS in VEGFR2 activation under altered GSH redox state. We believe a small decrease in GSH/GSSG would result in priming of VEGFR2 signaling pathway while a larger decrease in GSH/GSSG will result in dysfunctional signaling. In conclusion, the GSH/GSSH ratio is important in triggering and promoting VEGFR2 signaling.

Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling

Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H2O2 production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H2O2-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H2O2 generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H2O2- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H2O2-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.

20 - Endosomal Hydrogen Peroxide Production Leads to Localized Cysteine Sulfenic Acid Formation on Proteins during Lysophosphatidic Acid-Mediated Cell Signaling

20 - Endosomal Hydrogen Peroxide Production Leads to Localized Cysteine Sulfenic Acid Formation on Proteins during Lysophosphatidic Acid-Mediated Cell Signaling

Structure of the catalytic domain of protein tyrosine phosphatase sigma in the sulfenic acid form.

Protein tyrosine phosphatase sigma (PTPσ) plays a vital role in neural development. The extracellular domain of PTPσ binds to various proteoglycans, which control the activity of 2 intracellular PTP domains (D1 and D2). To understand the regulatory mechanism of PTPσ, we carried out structural and biochemical analyses of PTPσ D1D2. In the crystal structure analysis of a mutant form of D1D2 of PTPσ, we unexpectedly found that the catalytic cysteine of D1 is oxidized to cysteine sulfenic acid, while that of D2 remained in its reduced form, suggesting that D1 is more sensitive to oxidation than D2. This finding contrasts previous observations on PTPα. The cysteine sulfenic acid of D1 was further confirmed by immunoblot and mass spectrometric analyses. The stabilization of the cysteine sulfenic acid in the active site of PTP suggests that the formation of cysteine sulfenic acid may function as a stable intermediate during the redox-regulation of PTPs.

RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.

The reversible formation of cysteine sulfenic acid promotes B‐cell activation and proliferation

B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 min. Increased ROIs preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration-dependent block in anti-IgM-induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.

Mitochondrial superoxide mediates doxorubicin-induced keratinocyte apoptosis through oxidative modification of ERK and Bcl-2 ubiquitination

Massive apoptosis of keratinocytes has been implicated in the pathogenesis of chemotherapy-induced skin toxicities, but the underlying mechanisms of action are not well understood. The present study investigated the apoptotic effect of doxorubicin (DOX) on HaCaT keratinocytes and determined the underlying mechanisms. Treatment of the cells with DOX induced reactive oxygen species (ROS) generation and a concomitant increase in apoptotic cell death through the mitochondrial death pathway independent of p53. Electron spin resonance and flow cytometry studies showed that superoxide is the primary oxidative species induced by DOX and responsible for the death inducing effect. Ectopic expression of mitochondrial superoxide scavenging enzyme (MnSOD) or treatment with MnSOD mimetic (MnTBAP) inhibited DOX-induced superoxide generation and apoptosis. The mechanism by which superoxide mediates the apoptotic effect of DOX was shown to involve downregulation of Bcl-2 through ubiquitin–proteasomal degradation. Superoxide induces dephosphorylation of Bcl-2 through MAP kinase ERK1/2 inactivation, which promotes ubiquitination of Bcl-2. We also provide evidence for the oxidative modification of ERK1/2 through cysteine sulfenic acid formation. These findings indicate a novel pathway for redox regulation of apoptosis regulatory proteins, which could be important in the understanding of chemotherapy-induced toxicities and development of preventive treatment strategies which are currently lacking.

Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis

Reactive oxygen species (ROS) are important mediators for VEGF receptor 2 (VEGFR2) signalling involved in angiogenesis. The initial product of Cys oxidation, cysteine sulfenic acid (Cys-OH), is a key intermediate in redox signal transduction; however, its role in VEGF signalling is unknown. We have previously demonstrated IQGAP1 as a VEGFR2 binding scaffold protein involved in ROS-dependent EC migration and post-ischemic angiogenesis. Using a biotin-labelled Cys-OH trapping reagent, we show that VEGF increases protein-Cys-OH formation at the lamellipodial leading edge where it co-localizes with NADPH oxidase and IQGAP1 in migrating ECs, which is prevented by IQGAP1 siRNA or trapping of Cys-OH with dimedone. VEGF increases IQGAP1-Cys-OH formation, which is prevented by N-acetyl cysteine or dimedone, which inhibits VEGF-induced EC migration and capillary network formation. In vivo, hindlimb ischemia in mice increases Cys-OH formation in small vessels and IQGAP1 in ischemic tissues. In summary, VEGF stimulates localized formation of Cys-OH-IQGAP1 at the leading edge, thereby promoting directional EC migration, which may contribute to post-natal angiogenesis in vivo. Thus, targeting Cys-oxidized proteins at specific compartments may be the potential therapeutic strategy for various angiogenesis-dependent diseases.

The role of Reactive Oxygen Intermediates in B cell activation and differentiation (45.12)

Abstract Generating a protective humoral immune response requires proper B cell activation. Ligation of the BCR increases the level of reactive oxygen intermediates (ROI). While they have been implicated in oxidative stress, recent studies suggest ROI act as secondary messengers facilitating normal cellular function. Our focus is on the mechanisms by which ROI control B cell function. Following BCR ligation in vitro, the levels of the first oxidation product of cysteine, sulfenic acid, increased in the proteome. Using 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent further oxidation or reduction, we determined reversible cysteine sulfenic acid formation is critical for sustained tyrosine phosphorylation, calcium flux, and proliferation of B cells. To address the role of ROI in vivo, lymphocytic choriomeningitis virus (LCMV) infected mice were treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0-8 postinfection. On day 38, LCMV specific IgG antibody secreting cells (ASC) were 5-fold lower in the bone marrow. In contrast, the number of LCMV specific memory B cells was unaffected. The ASC decrease was not detected when mice were only treated from days 0-4 or 4-8 postinfection or during secondary viral infection. These data demonstrate ROI and reversible cysteine sulfenic acid formation are critical to B cell function and differentiation.

Formation of Cysteine Sulfenic Acid by Oxygen Atom Transfer from Nitrite

Cysteine sulfenic acid CysS(O)H is shown to be formed for the reaction of cysteine (CysSH) with aqueous nitrite and the water-soluble ferriheme models Fe(III)(TPPS) (TPPS = meso-tetra(4-sulfonatophenyl)porphyrinato) or Fe(III)(TMPS) (TMPS = meso-tetra(sulfonatomesityl)porphyrinato) at pH 5.8 and 7.4. The other product is the respective ferrous nitrosyl complex Fe(II)(Por)(NO) (Por = TPPS or TMPS). Analogous oxygen atom transfers (OAT) were seen when glutathione (GSH) was used as the substrate. The sulfenic acids, CysS(O)H and GS(O)H, are transient species since they react rapidly with excess thiol to give the respective disulfides, so their presence as reactive intermediates was demonstrated by trapping with dimedone and detecting the resulting adduct using LC/MS. Preliminary kinetics studies are consistent with rate-limiting OAT from a ferric nitro complex Fe(III)(Por)(NO(2)(-)) to CysSH, although this reaction is complicated by a competing dead-end equilibrium to form the thiolate complex (Fe(III)(TPPS)(CysS(-)).

The role of reactive oxygen intermediates (ROI) in B cell activation, proliferation, and differentiation (132.10)

Abstract Reactive oxygen intermediates (ROI) are generated during mitochondrial respiration, receptor ligation, and microorganism digestion. Ligation of the BCR resulted in ROI production that reaches a maximum by 15 minutes. ROI can modify all macromolecules, but we are interested in reversible oxidation events that modulate signaling. Our focus is on cysteine sulfenic acid, the first cysteine oxidation product. Using 5,5 dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent further oxidation or reduction, we addressed the role of reversible sulfenic acid formation in mature B cell activation, proliferation and differentiation. Following incubation with antibodies against surface bound Immunoglobulin M (IgM), mature B cells divide several times by three days. Incubation with dimedone results in a concentration dependent block of the initial division. This contrasted with dimedone-pretreated CpG DNA and LPS-stimulated B cells, where subsequent divisions were reduced compared to vehicle-treated cells. Dual stimulation with BAFF, CD40, and IL-4 did not rescue the effect of dimedone on proliferation. Cells pretreated with dimedone did not flux calcium when incubated with IgM, and in vitro plasma cell differentiation was also decreased. These studies demonstrate that mature B cell activation, proliferation and differentiation require reversible cysteine sulfenic acid formation.

Abstract 5329: ROS-dependent Cysteine Sulfenic Acid Formation of IQGAP1 and ERK1/2 in VEGF-induced Endothelial Cell Proliferation and Migration

VEGF receptor type 2 (VEGFR2) activation induces endothelial cell (EC) proliferation and migration. ROS-mediated protein modification involves oxidation of reactive cysteine residues to form a cysteine sulfenic acid (Cys-SOH) and subsequent oxidation products. However, role of Cys-SOH formation in VEGF signaling linked to angiogenesis is unknown. We previously identified IQGAP1, as a novel VEGFR2 binding protein, involved in regulating ROS-dependent EC migration and proliferation by recruiting Nox2 NADPH oxidase to activated VEGFR2 signaling complex. By using a newly-developed Cys-SOH trapping reagent we show that VEGF stimulation significantly increases protein-Cys-SOH formation in HUVECs. Immunofluorescence reveals that Cys-SOH modified proteins accumulate at the leading edge where they colocalize with NADPH oxidase, F-actin and IQGAP1 in actively migrating ECs. VEGF stimulation increases IQGAP1-Cys-SOH formation within 5min (60%) with a maximum increase at 15min (90%). VEGF-induced Cys-SOH formation of IQGAP1 is prevented by pretreatment with a GSH donor antioxidant, N-acetylcysteine. Knockdown of IQGAP1 by siRNA or trapping of Cys-SOH with dimedone, blocks wound-induced Cys-SOH formation at the leading edge and EC migration toward the injured site (80%), EC chemotaxis (90%) and matrigel angiogenesis (80%). These suggest linkage between IQGAP1-Cys-SOH formation and IQGAP1-mediated EC migration. In addition, dimedone pretreatment inhibits VEGF-stimulated EC proliferation. VEGF stimulation also induces oxidation of ERK1/2 upon 5 min (50%) and 15min (80%) stimulation. Trapping Cys-SOH by dimedone inhibits ERK phosphorylation (95%), which is associated with increasing IQGAP1/ERK complex (50%) after VEGF stimulation. Hind limb ischemia increases oxidation of tissue proteins including IQGAP1 (80%) and ERK1/2 (60%) at day 1 after injury, which is prevented in mice lacking Nox2. Thus, oxidation of IQGAP1 and ERK contributes to directional migration and proliferation of ECs and to neo-vascularization in ischemic tissue. The present study should provide insights into identifying and visualizing Cys-SOH formed proteins as important strategy for understanding mechanisms by which ROS regulate EC function such as angiogenesis.

Sulfenic Acid Modification: a Novel Link Between the Cardiovascular K+ Channel, Kv1.5, and Oxidative Stress

Atrial fibrillation, a life‐threatening arrhythmia, is strongly associated with oxidative stress. The voltage‐dependent potassium (Kv) channel, Kv1.5 is a prominent cardiovascular K+ channel that is vital for atrial repolarization. Numerous studies suggest that Kv channels are redox‐sensitive; however, a molecular link between oxidative stress and altered Kv1.5 expression/activity has not been established. Kv1.5 possesses intracellular cysteine residues that may be redox‐sensitive. Reversible formation of cysteine sulfenic acid in response to oxidative stress is a potentially important means of posttranslational protein regulation. Using a novel chemical probe, we find that Kv1.5 is modified with sulfenic acid in a redox‐sensitive manner. Mutagenesis to identify the redox‐sensitive cysteine residues reveals that sulfenic acid formation occurs on the COOH terminus. Using immunohistochemistry, we find that acute oxidant treatment decreases cell surface expression of Kv1.5, with no significant effect on Kv1.5 protein expression. Together, these data suggest that cellular redox state can induce formation of sulfenic acid and alter Kv1.5 cell surface density. Redox‐mediated alterations in cell surface density of ion channels may affect cellular excitability, leading to pro‐arrhythmic changes in cardiac action potentials. Research support: NIH Grants HL0270973 and T32ES07062.

Oxidation of archaeal peroxiredoxin involves a hypervalent sulfur intermediate

The oxidation of thiol groups in proteins is a common event in biochemical processes involving disulfide bond formation and in response to an increased level of reactive oxygen species. It has been widely accepted that the oxidation of a cysteine side chain is initiated by the formation of cysteine sulfenic acid (Cys-SOH). Here, we demonstrate a mechanism of thiol oxidation through a hypervalent sulfur intermediate by presenting crystallographic evidence from an archaeal peroxiredoxin (Prx), the thioredoxin peroxidase from Aeropyrum pernix K1 (ApTPx). The reaction of Prx, which is the reduction of a peroxide, depends on the redox active cysteine side chains. Oxidation by hydrogen peroxide converted the active site peroxidatic Cys-50 of ApTPx to a cysteine sulfenic acid derivative, followed by further oxidation to cysteine sulfinic and sulfonic acids. The crystal structure of the cysteine sulfenic acid derivative was refined to 1.77 A resolution with R(cryst) and R(free) values of 18.8% and 22.0%, respectively. The refined structure, together with quantum chemical calculations, revealed that the sulfenic acid derivative is a type of sulfurane, a hypervalent sulfur compound, and that the S(gamma) atom is covalently linked to the N(delta1) atom of the neighboring His-42. The reaction mechanism is revealed by the hydrogen bond network around the peroxidatic cysteine and the motion of the flexible loop covering the active site and by quantum chemical calculations. This study provides evidence that a hypervalent sulfur compound occupies an important position in biochemical processes.

The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8(+) T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8(+) T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8(+) and CD4(+) T cells. We also found that TNF-alpha production by effector and memory CD8(+) T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-gamma. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8(+) T cells are able to modulate signaling, proliferation, and function.

Fluorescent and Affinity-Based Tools To Detect Cysteine Sulfenic Acid Formation in Proteins

Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This species represents a biologically significant modification occurring during oxidant signaling or oxidative stress, and it can modulate protein function. Most methods to identify such oxidatively modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques ( Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. ( 2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjugate Chem . 16, 1624-1628 ). We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamine-, and three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification.

Characterization by Tandem Mass Spectrometry of Stable Cysteine Sulfenic Acid in a Cysteine Switch Peptide of Matrix Metalloproteinases

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.