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• W2000549663 abstract "Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana. Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana. Among the different amino acids, the sulfur containing amino acids like cysteine are particularly susceptible to oxidation by reactive oxygen species (ROS) [1]The abbreviations used are:ROSreactive oxygen speciesIAMiodoacetamideMMTSS-methyl methanethiosulfonateNEMN-ethylmaleimideSOHsulfenylation stateS-SdisulfidesSSGS-glutathionylationSNOS-nitrosothiolH2O2hydrogen peroxidePTMspost-translational modificationsc-CRDcarboxy-terminal cysteine-rich domainPAPperoxidase-anti-peroxidaseGOGene OntologyYAP1yeast AP-1 like [1]The abbreviations used are:ROSreactive oxygen speciesIAMiodoacetamideMMTSS-methyl methanethiosulfonateNEMN-ethylmaleimideSOHsulfenylation stateS-SdisulfidesSSGS-glutathionylationSNOS-nitrosothiolH2O2hydrogen peroxidePTMspost-translational modificationsc-CRDcarboxy-terminal cysteine-rich domainPAPperoxidase-anti-peroxidaseGOGene OntologyYAP1yeast AP-1 like (1Di Simplicio P. Franconi F. Frosalí S. Di Giuseppe D. Thiolation and nitrosation of cysteines in biological fluids and cells.Amino Acids. 2003; 25: 323-339Crossref PubMed Scopus (97) Google Scholar, 2Jacques S. Ghesquière B. Van Breusegem F. Gevaert K. Plant proteins under oxidative attack.Proteomics. 2013; 13: 932-940Crossref PubMed Scopus (51) Google Scholar). Recent studies suggest that the sulfenome, the initial oxidation products of cysteine residues, functions as an intermediate state of redox signaling (3Delaunay A. Pflieger D. Barrault M.-B. Vinh J. Toledano M.B. A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation.Cell. 2002; 111: 471-481Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, 4Tachibana T. Okazaki S. Murayama A. Naganuma A. Nomoto A. Kuge S. A major peroxiredoxin-induced activation of Yap1 transcription factor is mediated by reduction-sensitive disulfide bonds and reveals a low level of transcriptional activation.J. Biol. Chem. 2009; 284: 4464-4472Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Chiang S.M. Schellhorn H.E. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria.Arch. Biochem. Biophys. 2012; 525: 161-169Crossref PubMed Scopus (215) Google Scholar). Thus, identifying the sulfenome under oxidative stress is a way to detect potential redox sensors (6Leonard S.E. Reddie K.G. Carroll K.S. Mining the thiol proteome for sulfenic acid modifications reveals new targets for oxidation in cells.Acs. Chem. Biol. 2009; 4: 783-799Crossref PubMed Scopus (239) Google Scholar, 7Roos G. Messens J. Protein sulfenic acid formation: from cellular damage to redox regulation.Free Radic. Biol. Med. 2011; 51: 314-326Crossref PubMed Scopus (204) Google Scholar). reactive oxygen species iodoacetamide S-methyl methanethiosulfonate N-ethylmaleimide sulfenylation state disulfides S-glutathionylation S-nitrosothiol hydrogen peroxide post-translational modifications carboxy-terminal cysteine-rich domain peroxidase-anti-peroxidase Gene Ontology yeast AP-1 like reactive oxygen species iodoacetamide S-methyl methanethiosulfonate N-ethylmaleimide sulfenylation state disulfides S-glutathionylation S-nitrosothiol hydrogen peroxide post-translational modifications carboxy-terminal cysteine-rich domain peroxidase-anti-peroxidase Gene Ontology yeast AP-1 like This central role of the sulfenome in redox signaling provoked chemical biologists to develop strategies for sensitive detection and identification of sulfenylated proteins. The in situ trapping of the sulfenome is challenging because of two major factors: (1) the highly reactive, transient nature of sulfenic acids, which might be over-oxidized in excess of ROS, unless immediately protected by disulfide formation (7Roos G. Messens J. Protein sulfenic acid formation: from cellular damage to redox regulation.Free Radic. Biol. Med. 2011; 51: 314-326Crossref PubMed Scopus (204) Google Scholar); (2) the intracellular compartmentalization of the redox state that might be disrupted during extraction procedures, resulting in artificial non-native protein oxidations (8Go Y.-M. Jones D.P. Redox compartmentalization in eukaryotic cells.Biochim. Biophys. Acta. 2008; 1780: 1273-1290Crossref PubMed Scopus (502) Google Scholar, 9Leonard S.E. Carroll K.S. Chemical “omics” approaches for understanding protein cysteine oxidation in biology.Curr. Opin. Chem. Biol. 2011; 15: 88-102Crossref PubMed Scopus (148) Google Scholar). Having a sulfur oxidation state of zero, sulfenic acids can react as both electrophile and nucleophile, however, direct detection methods are based on the electrophilic character of sulfenic acid (10Gupta V. Carroll K.S. Sulfenic acid chemistry, detection, and cellular lifetime.Biochim. Biophys. Acta. 2014; 1840: 847-875Crossref PubMed Scopus (289) Google Scholar). In 1974, Allison and coworkers reported a condensation reaction between the electrophilic sulfenic acid and the nucleophile dimedone (5,5-dimethyl-1,3-cyclohexanedione), producing a corresponding thioether derivative (11Benitez L.V. Allison W.S. The inactivation of the acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase by dimedone and olefins.J. Biol. Chem. 1974; 249: 6234-6243Abstract Full Text PDF PubMed Google Scholar). This chemistry is highly selective and, since then, has been exploited to detect dimedone modified sulfenic acids using mass spectrometry (12Carballal S. Radi R. Kirk M.C. Barnes S. Freeman B.A. Alvarez B. Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite.Biochemistry. 2003; 42: 9906-9914Crossref PubMed Scopus (282) Google Scholar). However, dimedone has limited applications for cellular sulfenome identification because of the lack of a functional group to enrich the dimedone tagged sulfenic acids. Later, dimedone-biotin/fluorophores conjugates have been developed, which allowed sensitive detection and enrichment of sulfenic acid modified proteins (13Poole L.B. Klomsiri C. Knaggs S.A. Furdui C.M. Nelson K.J. Thomas M.J. Fetrow J.S. Daniel L.W. King S.B. Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins.Bioconjugate Chem. 2007; 18: 2004-2017Crossref PubMed Scopus (139) Google Scholar, 14Poole L.B. Zeng B.-B. Knaggs S.A. Yakubu M. King S.B. Synthesis of chemical probes to map sulfenic acid modifications on proteins.Bioconjugate Chem. 2005; 16: 1624-1628Crossref PubMed Scopus (114) Google Scholar, 15Charles R.L. Schröder E. May G. Free P. Gaffney P.R. Wait R. Begum S. Heads R.J. Eaton P. Protein sulfenation as a redox sensor – proteomics studies using a novel biotinylated dimedone analogue.Mol. Cell. Proteomics. 2007; 6: 1473-1484Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). This approach, however, was not always compatible with in vivo cellular sulfenome analysis, because the biotin/fluorophores-conjugated dimedone is membrane impermeable (9Leonard S.E. Carroll K.S. Chemical “omics” approaches for understanding protein cysteine oxidation in biology.Curr. Opin. Chem. Biol. 2011; 15: 88-102Crossref PubMed Scopus (148) Google Scholar) and endogenous biotinylated proteins might appear as false positives. More recently, the Carroll lab has developed DYn-2, a sulfenic acid specific chemical probe. This chemical probe consists of two functional units: a dimedone scaffold for sulfenic acid recognition and an alkyne chemical handle for enrichment of labeled proteins (9Leonard S.E. Carroll K.S. Chemical “omics” approaches for understanding protein cysteine oxidation in biology.Curr. Opin. Chem. Biol. 2011; 15: 88-102Crossref PubMed Scopus (148) Google Scholar). Once the sulfenic acids are tagged with the DYn-2 probe, they can be biotinylated through click chemistry (16Wang W. Hong S. Tran A. Jiang H. Triano R. Liu Y. Chen X. Wu P. Sulfated ligands for the copper(I)-catalyzed azide-alkyne cycloaddition.Chem. Asian J. 2011; 6: 2796-2802Crossref PubMed Scopus (81) Google Scholar). The click reaction used here is a copper (I)-catalyzed azide-alkyne cycloaddition reaction (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar), also known as azide-alkyne Huisgen cycloaddition (16Wang W. Hong S. Tran A. Jiang H. Triano R. Liu Y. Chen X. Wu P. Sulfated ligands for the copper(I)-catalyzed azide-alkyne cycloaddition.Chem. Asian J. 2011; 6: 2796-2802Crossref PubMed Scopus (81) Google Scholar). With this chemistry, a complex is formed between the alkyne functionalized DYn-2 and the azide functionalized biotin. This biotin functional group facilitates downstream detection, enrichment, and mass spectrometry based identification (Fig. 1). In an evaluation experiment, DYn-2 was found to efficiently detect H2O2-dependent sulfenic acid modifications in recombinant glutathione peroxidase 3 (Gpx3) of budding yeast (18Paulsen C.E. Truong T.H. Garcia F.J. Homann A. Gupta V. Leonard S.E. Carroll K.S. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity.Nat. Chem. Biol. 2012; 8: 57-64Crossref Scopus (342) Google Scholar). Moreover, it was reported that DYn-2 is membrane permeable, non-toxic, and a non-influencer of the intracellular redox balance (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar, 18Paulsen C.E. Truong T.H. Garcia F.J. Homann A. Gupta V. Leonard S.E. Carroll K.S. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity.Nat. Chem. Biol. 2012; 8: 57-64Crossref Scopus (342) Google Scholar). Therefore, DYn-2 has been suggested as a global sulfenome reader in living cells (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar, 18Paulsen C.E. Truong T.H. Garcia F.J. Homann A. Gupta V. Leonard S.E. Carroll K.S. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity.Nat. Chem. Biol. 2012; 8: 57-64Crossref Scopus (342) Google Scholar), and has been applied to investigate epidermal growth factor (EGF) mediated protein sulfenylation in a human epidermoid carcinoma A431 cell line and to identify intracellular protein targets of H2O2 during cell signaling (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar). Here, we selected the DYn-2 probe to identify the sulfenome in plant cells under oxidative stress. Through a combination of biochemical, immunoblot and mass spectrometry techniques, and TAIR10 database and SUBA3-software predictions, we can claim that DYn-2 is able to detect sulfenic acids on proteins located in different subcellular compartments of plant cells. We identified 226 sulfenylated proteins in response to an H2O2 treatment of Arabidopsis cell suspensions, of which 123 proteins are new candidates for cysteine oxidative post-translational modification (PTM) events. A. thaliana dark grown cell suspension line (PSB-D) was cultured as previously described (19Van Leene J. Stals H. Eeckhout D. Persiau G. Van De Slijke E. Van Isterdael G. De Clercq A. Bonnet E. Laukens K. Remmerie N. Henderickx K. De Vijlder T. Abdelkrim A. Pharazyn A. Van Onckelen H. Inzé D. Witters E. De Jaeger G. A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana.Mol. Cell. Proteomics. 2007; 6: 1226-1238Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). All experiments were performed with cells in mid-log phase (3-day old, around 10 mg fresh weight/ml). The time and dose of the stress treatment, as well as DYn-2 labeling were performed as follows: (1) For optimization of DYn-2 labeling conditions, we followed two conditions: (A) 10-ml cell cultures were stressed for 1 h by addition of 0, 0.1, 1 or 20 mm H2O2 in separated conical flasks (Merck, Germany). Then, the cells were harvested by filtration and rinsed with culture medium. After resuspension of the stressed cells in culture medium, probe labeling was performed with 0, 0.5, 1, 2.5, 5, or 10 mm of DYn-2 for 1 h. (B) The cell cultures were stressed for 1 h by addition of 0 or 20 mm H2O2 in the presence of 5 mm DYn-2. (2) For the detection of the dose-dependent responses of cells to H2O2 treatment, 10-ml cell cultures were treated with 0, 0.5, 1, 2, 5, 10, or 20 mm H2O2 in the presence of 500 μm DYn-2 for 1 h. For the detection of the time-dependent responses, 50-ml cell cultures were treated with 0, 1, or 20 mm H2O2 in the presence of 500 μm DYn-2. After 15, 30, 60, and 120 min treatment, 10 ml of cell culture were harvested at indicated time points for each H2O2 concentration. (3) For the competition study with the YAP1C probe, 10 ml of both YAP1C and YAP1A overexpressing Arabidopsis cell cultures were treated with 0 or 20 mm H2O2 for 1 h in the presence of 1 mm DYn-2 probe. For the optimization of DYn-2 labeling, the cells were treated with 20 mm H2O2 in the presence of 0, 0.5, 1, 2.5, 5, or 10 mm DYn-2 for 1 h. (4) For mass spectrometry based identification, 20-ml cell cultures were treated with 0 or 10 mm H2O2 for 30 min in the presence of 500 μm DYn-2. After stress treatment and DYn-2 probe labeling, the cells were harvested by filtration and washed 3 times with culture medium, then the cells were ready for protein extraction and click reaction following downstream analysis. Before each experiment, the concentration of H2O2 was determined at 240 nm using 43.6 m−1cm−1 as the molar extinction coefficient. For protein extraction and biotinylation by click reaction, we followed the protocol as previously described (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar) with some modifications. It is noteworthy to mention that the use of alkylating agents such as IAM and MMTS is not recommended, as they show reactivity with DYn-2 (unpublished data). Moreover, IAM, NEM, and MMTS are also known to form adducts with Cys-SOH, cleavable under reducing conditions (20Reisz J.A. Bechtold E. King S.B. Poole L.B. Furdui C.M. Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.FEBS J. 2013; 280: 6150-6161Crossref PubMed Scopus (81) Google Scholar). Harvested cells were ground on ice using sand with extraction buffer (25 mm Tris-HCl pH 7.6, 15 mm MgCl2, 150 mm NaCl, 15 mm pNO2PhenylPO4, 60 mm B-glycerolphosphate, 0.1% Nonidet P-40, 0.1 mm Na3VO4, 1 mm NaF, 1 mm phenylmethanesulfonyl fluoride, 1 μm E64, 1× Roche protease inhibitor mixture, 5% ethylene glycol) supplemented with catalase (bovine liver, Sigma-Aldrich, St Louis, MO) at 200 U/ml. The lysates were centrifuged at 16,100 × g for 30 min at 4 °C to remove the cell debris. The protein content from the soluble fractions was determined using a standard DC Protein Assay (Bio-Rad Laboratories Inc., Hercules, CA). After removing endogenous biotinylated proteins by NeutrAvidin agarose beads, a click reaction was performed with 100 μg of proteins for 1 h by a rocking incubation at room temperature (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar). By incubating for 5 min with 1 mm EDTA, the click reaction was stopped. Protein samples were denatured for 5 min at 96 °C, and then, 25 μg of each protein sample was resolved by SDS-PAGE. Sulfenylation was visualized by immunoblot with 1:80,000 dilution of streptavidin-HRP (Strep-HRP) antibody. Equal loading was confirmed on a Coomassie stained SDS-PAGE gel. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we performed the click reactions using 1-mg protein fractions after removing endogenous biotinylated proteins by NeutrAvidin agarose beads. Subsequently, the click reactions were stopped and proteins were precipitated in ice-cold acetone containing 10% trichloroacetic acid to remove nonreacted click reagents from the lysates upon incubation overnight at −20 °C. On the second day, the precipitated proteins were pelleted by centrifugation at 16,100 × g for 30 min at 4 °C. The pellet was washed twice with ice-cold acetone containing 5 mm dithiotreitol. Then, the pellet was air-dried to remove the acetone from the pellet. After complete resuspension of the precipitated proteins in PBS containing 0.2% SDS, the biotinylated DYn-2 labeled proteins were enriched with 200 μl Neutravidin agarose beads, which had been pre-equilibrated with resuspension buffer. The beads were collected by centrifugation at 2800 × g for 2 min, washed with PBS, which was followed by incubation with 5 mm dithiotreitol in the same buffer for 30 min at room temperature. Then, stringent washing steps were performed: 1× PBS, 1 × 1 m NaCl for 5 min, 1x PBS, 1 × 4 m urea for 5 min, 1× PBS, 1× PBS containing 0.2% (w/v) SDS, 3× PBS. After each washing step, the beads were collected by centrifugation as described above. The biotinylated proteins were eluted in 100 μl buffer solution containing 1 mm biotin in 50 mm Tris-HCl, pH 7.1, 1% SDS by boiling for 10 min. The eluted proteins were lyophilized and then resuspended in 15 μl/15 μl H2O/SDS loading buffer, resolved on SDS-PAGE as a single band (21Van Leene J. Eeckhout D. Cannoot B. De Winne N. Persiau G. Van De Slijke E. Vercruysse L. Dedecker M. Verkest A. Vandepoele K. Martens L. Witters E. Gevaert K. De Jaeger G. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.Nat. Protoc. 2015; 10: 169-187Crossref PubMed Scopus (95) Google Scholar), and excised for LC-MS/MS analysis. The gel bands were washed and subsequently digested in gel with trypsin. The obtained peptide mixtures were analyzed via LC-MS/MS using an Ultimate 3000 RSLC nano LC system (ThermoFisher Scientific, Bremen, Germany), in-line connected to a Q-Exactive mass spectrometer (ThermoFisher Scientific). Here, the peptides were first loaded on a trapping column (made in-house, 100 μm internal diameter (I.D.) × 20 mm, 5 μm beads C18 Reprosil-HD, Dr. Maisch, Ammerbuch-Entringen, Germany). After flushing from the trapping column, the sample was loaded on an analytical column (made in-house, 75 μm I.D. × 150 mm, 5 μm beads C18 Reprosil-HD, Dr. Maisch) packed in a needle (PicoFrit SELF/P PicoTip emitter, PF360-75-15-N-5, New Objective, Woburn, MA). Peptides were loaded with loading solvent (0.1% TFA in water/acetonitrile, 98/2 (v/v)) and separated using a linear gradient from 98% solvent A′ (0.1% formic acid in water) to 40% solvent B′ (0.1% formic acid in water/acetonitrile, 20/80 (v/v)) in 30 min at a flow rate of 300 nL/min. This is followed by a 5-min wash reaching 99% solvent B′. The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in a given MS spectrum. The source voltage was set at 3.4 kV and the capillary temperature was 275 °C. One MS1 scan (m/z 400–2000, AGC target 3 × 106 ions, maximum ion injection time 80 ms) acquired at a resolution of 70,000 (at 200 m/z) was followed by up to 10 tandem MS scans (resolution 17,500 at 200 m/z) of the most intense ions fulfilling predefined selection criteria (AGC target 5 × 104 ions, maximum ion injection time 60 ms, isolation window 2 Da, fixed first mass 140 m/z, spectrum data type: centroid, underfill ratio 2%, intensity threshold 1.7xE4, exclusion of unassigned, 1, 5–8, >8 charged precursors, peptide match preferred, exclude isotopes on, dynamic exclusion time 20 s). The HCD collision energy was set to 25% Normalized Collision Energy and the polydimethylcyclosiloxane background ion at 445.120025 Da was used for internal calibration (lock mass). MS/MS data in each LC run, Mascot Generic Files were created using the Distiller software (version 2.4.3.3, Matrix Science, www.matrixscience.com/Distiller). While generating these peak lists, grouping of spectra was allowed in Distiller with a maximal intermediate retention time of 30 s, and a maximum intermediate scan count of five was used where possible. Grouping was done with 0.005 Da precursor tolerances. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science, London, UK, www.matrixscience.com) using the Mascot Daemon interface (version 2.4, Matrix Science) against the TAIR10 database containing 35,386 protein sequences. The considered variable modifications were DYn-2-cycloaddition, oxidation, dioxidation, and trioxidation of the cysteine residues; oxidation of the methionine residues; pyro-glutamate formation of amino-terminal glutamine residues; and acetylation of the protein N terminus. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1), and on fragment ions to 20 mmu. The instrument setting was put on ESI-QUAD. Enzyme was set to trypsin, allowing for one missed cleavage, and cleavage was also allowed when lysine or arginine were followed by proline. Only peptides that were ranked first and scored above the threshold score, set at 99% confidence were withheld. Furthermore, we only included peptides with a minimum length of 8 residues and with a maximum mass deviation from the calculated mass of 2 ppm. The average PSM, peptide and protein FDRs for all analyses were calculated at 0.14%, 0.31% and 0.63% respectively, using the method of Käll et al. (22Käll L. Storey J.D. MacCoss M.J. Noble W.S. Posterior error probabilities and false discovery rates: two sides of the same coin.J. Proteome Res. 2008; 7: 40-44Crossref PubMed Scopus (210) Google Scholar). We considered the total unique identifications of two independent experimental rounds of the nontreated samples as the background dataset. For the data set of H2O2 treated samples, the overlapping identifications of three independent experiments were taken into account. To obtain the H2O2-dependent DYn-2 sulfenome, we subtracted the background data sets from the data set of the H2O2 treated identifications. For the labeling of sulfenylated proteins in living cells, it is of crucial importance to consider factors that might influence basal levels of cysteine oxidation (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar). For Arabidopsis cell suspension cultures, these factors could be the changes in physico-chemical parameters of the culture medium, nutrient deficiency, cells grown to the stationary phase, etc. We performed stress treatments with increasing concentrations of H2O2 on the 3-day-old PSB-D Arabidopsis cell suspension cultures in the presence of DYn-2 (Fig. 3 and supplemental Fig. S1A, 1B). After harvesting, cells were washed with culture medium to remove excess H2O2 and DYn-2. This washing step is necessary to avoid DYn-2 tagging of de novo sulfenylated proteins generated during the extraction process. Sample preparation and biotinylation of the DYn-2 tagged proteins with click chemistry were performed as previously described (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar), followed by protein separation on SDS-PAGE and visualization of the DYn-2 tagged biotinylated proteins on anti-Strep-HRP Western blots. We observed that DYn-2 is able to penetrate Arabidopsis cells and that it could detect sulfenic acids formed under stress. In contrast to mammalian cells (17Truong T.H. Carroll K.S. Bioorthogonal chemical reporters for analyzing protein sulfenylation in cells.Curr. Protoc. Chem. Biol. 2012; 4: 101-122Crossref Google Scholar), we found that the H2O2 stress treatment performed in the presence of the DYn-2 probe is an efficient approach to trap and visualize sulfenic acids in Arabidopsis cells (Fig. 3 and supplemental Fig. S1). Important to note is that we used a catalase-supplemented extraction buffer to extract soluble protein fractions. Catalase scavenges H2O2 that might be generated during the protein extraction procedure; in such a way we control de novo sulfenylation during the extraction. A pilot experiment using extraction buffer with and without catalase showed a clear influence of catalase to control post-extraction sulfenic acids formation at higher H2O2 concentrations (Fig. 3 and supplemental Fig. S2). By incubating the lysate with NeutrAvidin agarose beads, we removed endogenous biotinylated proteins and the nonsulfenylated proteins sticking to the beads. After optimizing the DYn-2 labeling conditions (H2O2 stress treatment in the presence of 500 μm DYn-2 probe (supplemental Fig. S1 and Fig. 3), we assessed whether DYn-2 interaction with sulfenylated proteins quantitatively affects the interaction of the YAP1C genetic probe with sulfenic acids under oxidative stress conditions. YAP1C is the carboxy-terminal, cysteine-rich domain (c-CRD) of the redox-regulated yeast AP-1 like (YAP1) transcription factor that has been adapted to trap protein sulfenic acids in vivo (23Takanishi C.L. Wood M.J. A genetically encoded probe for the identification of proteins that form sulfenic acid in response to H2O2 in Saccharomyces cerevisiae.J. Proteome Res. 2011; 10: 2715-2724Crossref PubMed Scopus (20) Google Scholar, 24Takanishi C.L. Ma L.-H. Wood M.J. A genetically encoded probe for cysteine sulfenic acid protein modification in vivo.Biochemistry. 2007; 46: 14725-14732Crossref PubMed Scopus (45) Google Scholar, 25Waszczak C. Akter S. Eeckhout D. Persiau G. Wahni K. Bodra N. Van Molle I. De Smet B. Vertommen D. Gevaert K. De Jaeger G. Van Montagu M. Messens J. Van Breusegem F. Sulfenome mining in Arabidopsis thaliana.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 11545-11550Crossref PubMed Scopus (130) Google Scholar). Briefly, we designed two variants of the YAP1 c-CRD: (1) YAP1C containing the wild-type redox regulatory Cys598 that traps CysSOH residues and (2) YAP1A, in which Cys598 is mutated to alanine as a control for nonspecific protein associations. YAP1 fragments were fused with a GS tag moiety for downstream analysis (26Van Leene J. Witters E. Inzé D. De Jaeger G. Boosting tandem affinity purification of plant protein complexes.Trends Plant Sci. 2008; 13: 517-520Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). With the help of a peroxidase-anti-peroxidase (PAP) antibody, which detects the GS tag moiety, we showed that in response to H2O2, YAP1C forms mixed disulfides with CysSOH proteins in an H2O2 concentration-dependent manner (25Waszczak C. Akter S. Eeckhout D. Persiau G. Wahni K. Bodra N. Van Molle I. De Smet B. Vertommen D. Gevaert K. De Jaeger G. Van Montagu M. Messens J. Van Breusegem F. Sulfenome mining in Arabidopsis thaliana.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 11545-11550Crossref PubMed Scopus (130) Google Scholar). However, these complexes were absent in YAP1A control cells, because the YAP1 c-CRD disulfide-bonded complexes are formed through the specific reaction of Cys598 with CysSOH on multiple proteins. We performed a competitive study between the DYn-2 and YAP1C probe. Therefore, the YAP1C and YAP1A overexpressing cells were stressed with 20 mm H2O2 for 1 h in the presence or absence of 1 mm DYn-2. As a control, we compared the response with nonstressed cells. Analysis of the Western blots with the PAP antibody showed that the intensity of YAP1C dimerization did not" @default.
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• W2000549663 date "2015-05-01" @default.
• W2000549663 modified "2023-10-14" @default.
• W2000549663 title "DYn-2 Based Identification of Arabidopsis Sulfenomes*" @default.
• W2000549663 cites W1525467602 @default.
• W2000549663 cites W1766401480 @default.
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