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• W1986320888 abstract "Redox-switches are critical cysteine thiols that are modified in response to changes in the cell’s environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT6, is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT6 quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches. Redox-switches are critical cysteine thiols that are modified in response to changes in the cell’s environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT6, is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT6 quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches. Changes in the oxidative balance can affect many aspects of cellular physiology through redox-signaling (1Hancock J.T. The role of redox mechanisms in cell signalling.Mol.Biotechnol. 2009; 2: 162-166Crossref Scopus (55) Google Scholar, 2Paulsen C.E. Carroll K.S. Orchestrating redox signaling networks through regulatory cysteine switches.Chem. Biol. 2010; 1: 47-62Google Scholar). Oxidative species modify critical cysteine thiols, known as redox-switches, which sense and respond to the cell’s fluctuating environment (3Georgiou G. How to flip the (redox) switch.Cell. 2002; 5: 607-610Abstract Full Text Full Text PDF Scopus (128) Google Scholar, 4Klomsiri C. Karplus P.A. Poole L.B. Cysteine-based redox switches in enzymes.Antioxid. Redox. Signal. 2011; 6: 1065-1077Crossref Scopus (274) Google Scholar). Depending on the magnitude, these fluctuations can affect normal metabolic processes, activate protective mechanisms or be cytotoxic. Redox-signaling is thought to derive from the integration of the type and concentration of oxidizing species, their associated chemical biology and cellular localization (5Winterbourn C.C. Hampton M.B. Thiol chemistry and specificity in redox signaling.Free Radic. Biol. Med. 2008; 5: 549-561Crossref Scopus (943) Google Scholar, 6Janssen-Heininger Y.M. Mossman B.T. Heintz N.H. Forman H.J. Kalyanaraman B. Finkel T. Stamler J.S. Rhee S.G. van der Vliet A. Redox-based regulation of signal transduction: principles, pitfalls, and promises.Free Radic. Biol. Med. 2008; 1: 1-17Crossref Scopus (628) Google Scholar, 7Thomas D.D. Ridnour L.A. Isenberg J.S. Flores-Santana W. Switzer C.H. Donzelli S. Hussain P. Vecoli C. Paolocci N. Ambs S. Colton C.A. Harris C.C. Roberts D.D. Wink D.A. The chemical biology of nitric oxide: implications in cellular signaling.Free Radic. Biol. Med. 2008; 1: 18-31Crossref Scopus (701) Google Scholar). However, less is known about the nature of the cysteine residues targeted or how oxidative signals are interpreted within the cell. S-nitrosylation (SNO), 1The abbreviations used are:AIF1apoptosis-inducing factor 1Biotin-HPDPN-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamidecysTMTcysteine reactive tandem mass tagEF2elongation factor 2GSHreduced glutathioneGSSGoxidized glutathioneGSNOS-nitrosoglutathioneHPAEChuman pulmonary arterial endothelia cellsL-NAMEL-NG-Nitroarginine methyl esterNEMN-ethylmaleimidePDBProtein DatabasePRIDEproteomics identifications databasePrxperoxiredoxinsSNOS-nitrosylationSNO-CysS-nitrosocysteine. 1The abbreviations used are:AIF1apoptosis-inducing factor 1Biotin-HPDPN-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamidecysTMTcysteine reactive tandem mass tagEF2elongation factor 2GSHreduced glutathioneGSSGoxidized glutathioneGSNOS-nitrosoglutathioneHPAEChuman pulmonary arterial endothelia cellsL-NAMEL-NG-Nitroarginine methyl esterNEMN-ethylmaleimidePDBProtein DatabasePRIDEproteomics identifications databasePrxperoxiredoxinsSNOS-nitrosylationSNO-CysS-nitrosocysteine. also known as S-nitrosation, is emerging as an important regulatory post-translational modification in many cellular processes (8Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 2: 150-166Crossref Scopus (1722) Google Scholar). This modification is the result of the covalent addition of an NO group to a cysteine thiol; however, the specific mechanism of this addition has not been fully determined (9Forman H.J. Fukuto J.M. Torres M. Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers.Am. J. Physiol. Cell Physiol. 2004; 2: C246-C256Crossref Scopus (455) Google Scholar). SNO possesses the essential criteria for a signaling modification including a rapid reaction, specificity and enzymatic reduction (10Benhar M. Forrester M.T. Hess D.T. Stamler J.S. Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins.Science. 2008; 5879: 1050-1054Crossref Scopus (451) Google Scholar). SNO has been associated with a variety of diseases making it the subject of intensifying research interest (8Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 2: 150-166Crossref Scopus (1722) Google Scholar). Nitric oxide stimulation has been found to generate a multitude of biological responses from protective to cytotoxic which can be stratified based on concentration (7Thomas D.D. Ridnour L.A. Isenberg J.S. Flores-Santana W. Switzer C.H. Donzelli S. Hussain P. Vecoli C. Paolocci N. Ambs S. Colton C.A. Harris C.C. Roberts D.D. Wink D.A. The chemical biology of nitric oxide: implications in cellular signaling.Free Radic. Biol. Med. 2008; 1: 18-31Crossref Scopus (701) Google Scholar) suggesting that the reactivity of specific redox-switches may play a role in regulating these effects. apoptosis-inducing factor 1 N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide cysteine reactive tandem mass tag elongation factor 2 reduced glutathione oxidized glutathione S-nitrosoglutathione human pulmonary arterial endothelia cells L-NG-Nitroarginine methyl ester N-ethylmaleimide Protein Database proteomics identifications database peroxiredoxins S-nitrosylation S-nitrosocysteine. apoptosis-inducing factor 1 N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide cysteine reactive tandem mass tag elongation factor 2 reduced glutathione oxidized glutathione S-nitrosoglutathione human pulmonary arterial endothelia cells L-NG-Nitroarginine methyl ester N-ethylmaleimide Protein Database proteomics identifications database peroxiredoxins S-nitrosylation S-nitrosocysteine. Because of their labile nature, SNO-modifications can be difficult to study with traditional biochemical techniques. In 2001, Jaffrey and Snyder introduced the biotin switch assay which utilizes a replacement strategy to stably label SNO-modified cysteines allowing their detection and identification (11Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide.Nat. Cell. Biol. 2001; 2: 193-197Crossref Scopus (1222) Google Scholar, 12Jaffrey S.R. Snyder S.H. The biotin switch method for the detection of S-nitrosylated proteins.Sci STKE. 2001; 86: pl1Google Scholar). Replacement is achieved by first blocking free thiols with an alkylating agent then reducing SNO-modifications with ascorbate and labeling with a thiol-reactive biotin or resin. These reagents form mixed disulfide bonds with the previously SNO-modified thiols (Fig. 1A). Once labeled, biotinylated proteins can be easily detected or enriched with streptavidin. Labeled peptides can also be captured and analyzed by MS, providing large-scale SNO-site identification (13Lam Y.W. Yuan Y. Isaac J. Babu C.V. Meller J. Ho S.M. Comprehensive identification and modified-site mapping of S-nitrosylated targets in prostate epithelial cells.PLoS One. 2010; 2: e9075Crossref Scopus (74) Google Scholar, 14Hao G. Derakhshan B. Shi L. Campagne F. Gross S.S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.Proc. Natl. Acad. Sci. U.S.A. 2006; 4: 1012-1017Crossref Scopus (298) Google Scholar). Despite the popularity of this technique, limitations have been identified which have been reviewed in (15Foster M.W. Methodologies for the characterization, identification and quantification of S-nitrosylated proteins.Biochim. Biophys. Acta. 2011; 10.1016/j.bbagen.2011.03.013Google Scholar, 16Forrester M.T. Foster M.W. Benhar M. Stamler J.S. Detection of protein S-nitrosylation with the biotin-switch technique.Free Radic. Biol. Med. 2009; 2: 119-126Crossref Scopus (241) Google Scholar). Common critiques include the use of ascorbate as the specific reducing agent which is suspected of reducing disulfide bonds or other oxidiative modifications and the lack of a permanent label at the modified cysteine residue that is detectable by MS analysis which can lead to ambiguity in site identifications. Each of these concerns has the potential to increase the incidence of false-positive results. Different variations of the assay offer several accommodations for these issues (13Lam Y.W. Yuan Y. Isaac J. Babu C.V. Meller J. Ho S.M. Comprehensive identification and modified-site mapping of S-nitrosylated targets in prostate epithelial cells.PLoS One. 2010; 2: e9075Crossref Scopus (74) Google Scholar, 17Huang B. Chen C. Detection of protein S-nitrosation using irreversible biotinylation procedures (IBP).Free Radic. Biol. Med. 2010; 3: 447-456Crossref Scopus (52) Google Scholar, 18Whalen E.J. Foster M.W. Matsumoto A. Ozawa K. Violin J.D. Que L.G. Nelson C.D. Benhar M. Keys J.R. Rockman H.A. Koch W.J. Daaka Y. Lefkowitz R.J. Stamler J.S. Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2.Cell. 2007; 3: 511-522Abstract Full Text Full Text PDF Scopus (253) Google Scholar, 19Murray C.I. Kane L.A. Uhrigshardt H. Wang S.B. Van Eyk J.E. Site-mapping of in vitro S-nitrosation in cardiac mitochondria: implications for cardioprotection.Mol.Cell. Proteomics. 2011; 3 (M110 004721)Google Scholar, 20Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 12: 1154-1169Abstract Full Text Full Text PDF Scopus (3680) Google Scholar, 21Forrester M.T. Thompson J.W. Foster M.W. Nogueira L. Moseley M.A. Stamler J.S. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.Nat. Biotechnol. 2009; 6: 557-559Crossref Scopus (307) Google Scholar, 22Paige J.S. Xu G. Stancevic B. Jaffrey S.R. Nitrosothiol reactivity profiling identifies S-nitrosylated proteins with unexpected stability.Chem. Biol. 2008; 12: 1307-1316Abstract Full Text Full Text PDF Scopus (149) Google Scholar); however, there is currently no unified solution. Here, we present a new approach to the biotin switch assay using the cysteine reactive tandem mass tag (cysTMT) reagent to specifically detect, identify, and quantify SNO-modified sites. CysTMT6 is a thiol reactive version of tandem mass tag that has been established for multiplex mass spectrometry analysis (23Thompson A. Schafer J. Kuhn K. Kienle S. Schwarz J. Schmidt G. Neumann T. Johnstone R. Mohammed A.K. Hamon C. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.Anal. Chem. 2003; 8: 1895-1904Crossref Scopus (1709) Google Scholar). This new reagent fulfills the requirements for a biotin switch label and offers some distinct advantages, including a permanent mass tag and the fragmentation of up to 6 isotopically balanced reporter ions between 126–131 Da permitting multiplex quantification. Using this technique we demonstrate specific detection of SNO-modified sites and quantify the response of individual cysteine residues to GSNO treatment by mapping the continuum of protein thiol-reactivity to SNO-modification. cysTMT6, cysTMT0, N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide (biotin-HPDP), TMT affinity resin, Streptavidin Plus UltraLink Resin and Zeba desalt spin columns were from Thermo Fisher Scientific (www.thermofisher.com). Stock solutions of S-nitrocysteine (SNO-Cys) were prepared fresh before each experiment according to the protocol outlined by Park and Kosta (24Park J.K. Kostka P. Fluorometric detection of biological S-nitrosothiols.Anal. Biochem. 1997; 1: 61-66Crossref Scopus (53) Google Scholar) scaled up 10 fold. Prepared solutions were found to contain 6–7 mm SNO-Cys. All other reagents including S-nitrosoglutathione (GSNO) and other chemicals were obtained from Sigma-Aldrich (www.sigmaaldrich.com). Human pulmonary arterial endothelia cells (HPAEC) (Lonza, www.lonza.com) were grown in EGM®-2 Endothelial Cell Growth Medium-2 (Lonza). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and used between passages 6 and 9. Proteins were separated by SDS-PAGE NuPage 4–12%, 1-mm gels (Invitrogen, www.invitrogen.com) with 2-(N-morpholino)ethanesulfonic acid running buffer. Proteins were transferred to nitrocellulose membranes which were blocked for 1 h with 5% (w/v) nonfat milk powder in TBS-t (50 mm Tris-HCl pH 7.4, 150 mm NaCl, 0.1% (v/v) tween 20). CysTMT labeled proteins were detected by incubating with anti-TMT primary antibody (Thermo Fisher Scientific) followed by anti-mouse alkaline phosphatase conjugated secondary antibody (Jackson ImmunoResearch Inc., www.jacksonimmuno.com) in blocking solution. Biotin labeled proteins were detected by incubation with streptavidin conjugated alkaline phosphatase (Jackson ImmunoResearch Inc.). Blots were washed overnight at 4 °C and developed using immun-star substrate (Bio-Rad, www.Bio-Rad.com). SNO-modifications were detected in GSNO treated HPAEC lysates using the biotin switch assay (12Jaffrey S.R. Snyder S.H. The biotin switch method for the detection of S-nitrosylated proteins.Sci STKE. 2001; 86: pl1Google Scholar, 14Hao G. Derakhshan B. Shi L. Campagne F. Gross S.S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.Proc. Natl. Acad. Sci. U.S.A. 2006; 4: 1012-1017Crossref Scopus (298) Google Scholar) (discussed in (14Hao G. Derakhshan B. Shi L. Campagne F. Gross S.S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.Proc. Natl. Acad. Sci. U.S.A. 2006; 4: 1012-1017Crossref Scopus (298) Google Scholar, 16Forrester M.T. Foster M.W. Benhar M. Stamler J.S. Detection of protein S-nitrosylation with the biotin-switch technique.Free Radic. Biol. Med. 2009; 2: 119-126Crossref Scopus (241) Google Scholar, 25Derakhshan B. Wille P.C. Gross S.S. Unbiased identification of cysteine S-nitrosylation sites on proteins.Nat. Protoc. 2007; 7: 1685-1691Crossref Scopus (83) Google Scholar), see Fig. 1A for reaction schema). HPAECs were lysed in 20 mm HEPES pH 7.4, 150 mm NaCl, 1 mm EDTA containing 1.0% (v/v) triton X-100 using a probe sonicator and centrifuged for 10 min at 2000 × g. The protein concentration of the resulting supernatant was determined by bicinchoninic acid assay and diluted to 0.8 μg/μl in HEN (250 mm HEPES pH 7.4, 1 mm EDTA and 0.1 mm neocuproine) including 0.4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate. 200 μg of cell lysate were treated with 2, 10 or 20 μm GSNO or three different control treatments (untreated vehicle, 20 μm reduced glutathione (GSH) or 10 μm oxidized glutathione (GSSG)) for 15 min at 37 °C (n = 3). GSNO is commonly used as the NO-donor for in vitro investigations due to its relative ease of use and because small thiol-containing molecules including glutathione are thought to play an important role in intracellular SNO-modification (8Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 2: 150-166Crossref Scopus (1722) Google Scholar, 26Irwin C. Roberts W. Naseem K.M. Nitric oxide inhibits platelet adhesion to collagen through cGMP-dependent and independent mechanisms: the potential role for S-nitrosylation.Platelets. 2009; 7: 478-486Crossref Scopus (44) Google Scholar, 27Lima B. Lam G.K. Xie L. Diesen D.L. Villamizar N. Nienaber J. Messina E. Bowles D. Kontos C.D. Hare J.M. Stamler J.S. Rockman H.A. Endogenous S-nitrosothiols protect against myocardial injury.Proc. Natl. Acad. Sci. U.S.A. 2009; 15: 6297-6302Crossref Scopus (191) Google Scholar, 28Marino S.M. Gladyshev V.N. Structural analysis of cysteine S-nitrosylation: a modified acid-based motif and the emerging role of trans-nitrosylation.J. Mol. Biol. 2010; 4: 844-859Crossref Scopus (172) Google Scholar, 29Han P. Chen C. Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins.Rapid Commun. Mass Spectrom. 2008; 8: 1137-1145Crossref Scopus (40) Google Scholar). To control for the possibility that the glutathione carrier may modify some cysteine thiols, reduced and oxidized glutathione were used as donor controls. All steps were performed in the dark or protected from light. Treatment compounds were removed using an HEN equilibrated Zeba desalt spin column according to the manufacturer’s protocol. The remaining free thiols were blocked with 20 mm N-ethylmaleimide (NEM) in the presence of 2.5% (w/v) SDS and incubated for 20 min at 50 °C. Excess NEM was removed by acetone precipitation. As a positive control, an additional untreated sample was processed but not blocked with NEM. This allowed for the labeling of all the endogenously available cysteine residues in the lysate. SNO-modified thiols were reduced and labeled by resuspending pellets in HENS (HEN containing 1.0% (w/v) SDS), 1 mm ascorbate, 1 mm CuSO4 and 0.8 mm Biotin-HPDP, cysTMT6 or, in the case of the positive controls, cysTMT0 reagent and incubated at room temp for 2 h. CuSO4 was included as it has been found to increase the sensitivity but not affect specificity of the ascorbate reduction of SNO-modifications (30Wang X. Kettenhofen N.J. Shiva S. Hogg N. Gladwin M.T. Copper dependence of the biotin switch assay: modified assay for measuring cellular and blood nitrosated proteins.Free Radic. Biol. Med. 2008; 7: 1362-1372Crossref Scopus (92) Google Scholar). TMT samples were labeled with cysTMT126–131 in the order; untreated, GSH, GSSG, 2, 10 and 20 μm GSNO for two of the replicates and labeled in reverse order for the third. Data obtained from reverse TMT labeling have been reordered in the data table for ease of analysis. Excess label was removed by acetone precipitation (2 volumes) and the resultant pellets were carefully washed with an additional volume of acetone. Pellets were resuspended to 5 μg/μl with HENS. For gel electrophoresis analysis, 200 μg of biotin-HPDP labeled protein were diluted 20 fold in neutralization buffer (20 mm HEPES, 150 mm, NaCl 1 mm EDTA, 0.5% (v/v) triton X-100). Biotinylated proteins were captured by incubation with 15 μl of washed, packed ultralink immobilized streptavidin beads for 1 h at room temperature. Beads were washed with 4 × 50 bead volumes of wash buffer (20 mm HEPES, 600 mm NaCl 1 mm EDTA, 0.5% (v/v) triton X-100) and 2 × with elution buffer (20 mm HEPES pH 7.7, 100 mm NaCl, 1 mm EDTA). Captured proteins were eluted with 40 μl of elution buffer containing 100 mm dithiotreitol. Eluted samples were mixed with 15 μl of 4 × LDS sample buffer, boiled, separated by SDS-PAGE and silver stained according to the protocol described in (31Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 5: 850-858Crossref Scopus (7807) Google Scholar). For MS studies and quantification, 200 μg of each cysTMT6 sample were combined (1200 μg total), diluted to 1.8 ml with TBS pH 7.0, passed through a Zeba desalt spin column equilibrated with TBS. The resulting samples were adjusted to 0.02% (v/v) RapigestTM SF (Waters, www.waters.com) and digested overnight with 30 μg of trypsin (Promega, www.promega.com) at 37 °C. Digestions were halted by 0.25 mm phenylmethylsulfonyl fluoride and then TMT labeled peptides were captured by 2 h incubation with 600 μl of TMT affinity resin slurry at room temperature. Unlabeled peptides were removed by washing with 3 × 5 ml of TBS, 3 × 5 ml TBS containing 4 m urea followed by 3 × 5 ml of TBS. Beads were then incubated for 2 h at room temperature followed by 3 × 5 ml washes of TBS and then 2 × 5 ml ddH2O to reduce buffering capacity and salt content. CysTMT labeled peptides were eluted with 2 × 600 μl portions of 50% (v/v) ACN/0.4% (v/v) trifluoroacetic acid and combined. Samples were dried in a speed vac and then cleaned using a detergent removal spin column (Thermo Fisher Scientific) and C18 UltraMicroSpin™ column (Nest Group, www.nestgrp.com) both according to the manufacturer’s protocol. Peptide identification by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis was performed using an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) interfaced with a NanoAquity UPLC system (Waters, www.waters.com) using Xcalibur 2.1. Peptides were loaded on to a hand packed column consisting of 75 μm x 15 cm of Michrom Magic C18 (5 μm particles with 100 Å pore size) at 750 nL/min for 15 min at 3% B (B = 90% (v/v) ACN 0.1% (v/v) formic acid, A = 0.1% (v/v) formic acid). Elution was performed at 300 nl/min by increasing the gradient to 55% B over 95 min before a bump to 100% B for 4 min followed by and re-equilibration back to 3% B for 10 min. Precursors were acquired at 30,000 resolution and 15,000 for the fragment ions with a target of 1e6 and 1e5 for full scan and MS/MS respectively. Isolation width was set to 1.2 daltons and normalized collision energy was set to 45. HCD was used exclusively with a lock mass of the polysiloxane at 371.101230 for MS1 and MS2. Xcalibur raw files were converted to mzXML files using pwiz (pwiz-bin-windows-x86-vc90-release-2_0_1905). MS data were searched against the International Protein Index human primary sequence database (version v3.62, 83947 entries) (32Kersey P.J. Duarte J. Williams A. Karavidopoulou Y. Birney E. Apweiler R. The International Protein Index: an integrated database for proteomics experiments.Proteomics. 2004; 7: 1985-1988Crossref Scopus (640) Google Scholar) using Sorcerer 2™-SEQUEST® (version v.27, rev. 11) (Sage-N Research, www.sagenresearch.com) with post search analysis performed using the trans-proteome pipeline and Scaffold 3 (Proteome Software Inc. www.proteomesoftware.com) implementing the PeptideProphet (33Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 20: 5383-5392Crossref Scopus (3886) Google Scholar) and ProteinProphet (34Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 17: 4646-4658Crossref Scopus (3621) Google Scholar) algorithms. All raw data peak extraction was performed using the Sorcerer 2™-SEQUEST® default settings. Search parameters included semi-enzyme digest with trypsin (after Arg or Lys) with up to 2 missed cleavages. SEQUEST® was searched with a parent ion tolerance of 10 ppm and a fragment ion mass tolerance of 1.00 Da with the dynamic modifications to NEM (C), cysTMT6 or cysTMT0 (C), oxidation (M). Peptide identifications were accepted if they had an Xcorr score greater than 2.2 and a probability greater than 95.0% as specified by the PeptideProphet algorithm (33Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 20: 5383-5392Crossref Scopus (3886) Google Scholar). Protein identifications were accepted if they could be established at greater than 95.0% probability as assigned by the ProteinProphet algorithm and contained at least 1 identified peptide (34Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 17: 4646-4658Crossref Scopus (3621) Google Scholar). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. False discovery rates were calculated by the ProteinProphet algorithm (34Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 17: 4646-4658Crossref Scopus (3621) Google Scholar). Quantitative values for the TMT reporter ions were collected with the Libra module of the trans-proteome pipeline using a custom condition file (see online supplement). The data presented in supplemental Tables S1 and S3 contains only those peptides containing a cysTMT6 modification. The position of each cysTMT6-modified cysteine was determined by mapping the peptide against the full protein sequence. For analysis, reporter ions were normalized to total reporter ion signal within each spectrum and then values were averaged across all peptide observations for an individual site. A site of SNO-modification was determined if the average 20 μm GSNO signal was at least twofold greater than the average of the largest control condition (untreated, 20 μm GSH, 10 μm GSSG). For subsequent analysis, the background (untreated) signal was subtracted from each of the other conditions. The extent of potential NO-carrier modification was assessed by determining the extent of either GSH or GSSG signal compared with 20 μm GSNO. To determine the response of each individual cysteine to the NO-donor, a slope for the SNO-response to 2, 10, and 20 μm GSNO was calculated by nonlinear regression. Cysteine residues with a slope less than 0.9 were considered to be sensitive, between 0.9 and 1.1 (1.0 ± 10%) displayed a linear response and greater than 1.1 were reactive but considered less sensitive to the NO-donor. Nonlinear regression was not performed for the most insensitive sites, where signal was only detected in the 20 μm GSNO, instead a reactivity value of 3.5 was assigned. Twenty-five target cysteine residues were selected from each of the response groups and 20 residues on either side of each site were submitted for consensus sequence analysis. After alignment with ClustalW2 (www.ebi.ac.uk/Tools/clustalw2/index.html), the sequences were screened for conserved motifs using the CONSENSUS, TEIRESIAS, and PRATT algorithms (http://coot.embl.de/Alignment/consensus.html; http://cbcsrv.watson.ibm.com/Tspd.html; www.ebi.ac.uk/Tools/pratt/index.html), respectively (35Rigoutsos I. Floratos A. Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm.Bioinformatics. 1998; 1: 55-67Crossref Scopus (406) Google Scholar). Frequencies of fla" @default.
• W1986320888 created "2016-06-24" @default.
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• W1986320888 date "2012-02-01" @default.
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• W1986320888 title "Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay" @default.
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