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• W1997455293 abstract "Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angeli's salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS3 analysis. These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angeli's salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets. Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angeli's salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS3 analysis. These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angeli's salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; AS, Angeli's salt; CF, correction factor; A, average fragment peak area; ECD, electron capture dissociation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNO, nitroxyl; MRM, multiple reaction monitoring; nESI, nano-ESI; 1D, one-dimensional; Qq-TOF, quadrupole Q-TOF; LTQ, linear ion trap quadrupole. has emerged as an important physiological signaling molecule, particularly in the vascular, neuronal, and immune systems. NO regulates many processes including platelet function, vascular tone, and leukocyte recruitment mainly through the cGMP second messenger system (1Moncada S. Palmer R.M. Higgs E.A. Nitric oxide: physiology, pathophysiology, and pharmacology.Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). More recent studies have shown that nitric oxide can react directly with a number of different biological species including metal centers of proteins, nucleophilic amino acid residues (nitrosation/S-nitrosylation), and aromatic amino acid residues (nitration) (2Ischiropoulos H. Gow A. Pathophysiological functions of nitric oxide-mediated protein modifications.Toxicology. 2005; 208: 299-303Crossref PubMed Scopus (69) Google Scholar, 3Hurd T.R. Filipovska A. Costa N.J. Dahm C.C. Murphy M.P. Disulphide formation on mitochondrial protein thiols.Biochem. Soc. Trans. 2005; 33: 1390-1393Crossref PubMed Scopus (65) Google Scholar), and the products of these reactions have been analyzed by mass spectrometry (4Hao G. Gross S.S. Electrospray tandem mass spectrometry analysis of S- and N-nitrosopeptides: facile loss of NO and radical-induced fragmentation.J. Am. Soc. Mass Spectrom. 2006; 17: 1725-1730Crossref PubMed Scopus (81) Google Scholar, 5Greis K.D. Zhu S. Matalon S. Identification of nitration sites on surfactant protein A by tandem electrospray mass spectrometry.Arch. Biochem. Biophys. 1996; 335: 396-402Crossref PubMed Scopus (62) Google Scholar, 6Sarver A. Scheffler N.K. Shetlar M.D. Gibson B.W. Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometry.J. Am. Soc. Mass Spectrom. 2001; 12: 439-448Crossref PubMed Scopus (134) Google Scholar, 7Petersson A.S. Steen H. Kalume D.E. Caidahl K. Roepstorff P. Investigation of tyrosine nitration in proteins by mass spectrometry.J. Mass Spectrom. 2001; 36: 616-625Crossref PubMed Scopus (134) Google Scholar, 8Gorman J.J. Wallis T.P. Pitt J.J. Protein disulfide bond determination by mass spectrometry.Mass. Spectrom. Rev. 2002; 21: 183-216Crossref PubMed Scopus (225) Google Scholar). The biological relevance of these reactions is slowly coming to light, and NO-mediated S-nitrosylation has now been linked to a number of diseases including diabetes, multiple sclerosis, cystic fibrosis, and asthma (9Foster M.W. McMahon T.J. Stamler J.S. S-Nitrosylation in health and disease.Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). Nitroxyl (HNO/NO−), an alternative redox form of NO, has only recently begun to draw attention in the biomedical research community. The interest associated with HNO is due to its novel and important biological activity (10Paolocci N. Saavedra W.F. Miranda K.M. Martignani C. Isoda T. Hare J.M. Espey M.G. Fukuto J.M. Feelisch M. Wink D.A. Kass D.A. Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10463-10468Crossref PubMed Scopus (260) Google Scholar, 11Paolocci N. Katori T. Champion H.C. St John M.E. Miranda K.M. Fukuto J.M. Wink D.A. Kass D.A. Positive inotropic and lusitropic effects of HNO/NO− in failing hearts: independence from β-adrenergic signaling..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5537-5542Crossref PubMed Scopus (282) Google Scholar, 12Bermejo E. Saenz D.A. Alberto F. Rosenstein R.E. Bari S.E. Lazzari M.A. Effect of nitroxyl on human platelets function.Thromb. Haemostasis. 2005; 94: 578-584Crossref PubMed Scopus (50) Google Scholar, 13Colton C.A. Gbadegesin M. Wink D.A. Miranda K.M. Espey M.G. Vicini S. Nitroxyl anion regulation of the NMDA receptor.J. Neurochem. 2001; 78: 1126-1134Crossref PubMed Scopus (43) Google Scholar, 14Lee M.J. Nagasawa H.T. Elberling J.A. DeMaster E.G. Prodrugs of nitroxyl as inhibitors of aldehyde dehydrogenase.J. Med. Chem. 1992; 35: 3648-3652Crossref PubMed Scopus (61) Google Scholar, 15Norris A.J. Sartippour M.R. Lu M. Park T. Rao J.Y. Jackson M.I. Fukuto J.M. Brooks M.N. Nitroxyl inhibits breast tumor growth and angiogenesis.Int. J. Cancer. 2008; 122: 1905-1910Crossref PubMed Scopus (78) Google Scholar). There has been particular interest in the effect of HNO on failing hearts as it has been shown to increase left ventricular contractility and, at the same time, to lower cardiac preload and diastolic pressure without increasing arterial resistance (10Paolocci N. Saavedra W.F. Miranda K.M. Martignani C. Isoda T. Hare J.M. Espey M.G. Fukuto J.M. Feelisch M. Wink D.A. Kass D.A. Nitroxyl anion exerts redox-sensitive positive cardiac inotropy in vivo by calcitonin gene-related peptide signaling.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10463-10468Crossref PubMed Scopus (260) Google Scholar, 11Paolocci N. Katori T. Champion H.C. St John M.E. Miranda K.M. Fukuto J.M. Wink D.A. Kass D.A. Positive inotropic and lusitropic effects of HNO/NO− in failing hearts: independence from β-adrenergic signaling..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5537-5542Crossref PubMed Scopus (282) Google Scholar), effects that indicate the potential for HNO to be developed as a treatment for heart failure (16Feelisch M. Nitroxyl gets to the heart of the matter.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4978-4980Crossref PubMed Scopus (73) Google Scholar). As well, treatment of platelets with micromolar concentrations of the HNO donor Angeli's salt (AS) leads to an inhibition of platelet aggregation that was found to be both time- and dose-dependent (12Bermejo E. Saenz D.A. Alberto F. Rosenstein R.E. Bari S.E. Lazzari M.A. Effect of nitroxyl on human platelets function.Thromb. Haemostasis. 2005; 94: 578-584Crossref PubMed Scopus (50) Google Scholar). Other pharmacological studies have shown that HNO can be protective against excitotoxicity of the N-methyl-d-aspartate receptor (13Colton C.A. Gbadegesin M. Wink D.A. Miranda K.M. Espey M.G. Vicini S. Nitroxyl anion regulation of the NMDA receptor.J. Neurochem. 2001; 78: 1126-1134Crossref PubMed Scopus (43) Google Scholar, 17Kim W.K. Choi Y.B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Attenuation of NMDA receptor activity and neurotoxicity by nitroxyl anion, NO−.Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar); it can inhibit aldehyde dehydrogenase, which could be used as an antialcoholic treatment (14Lee M.J. Nagasawa H.T. Elberling J.A. DeMaster E.G. Prodrugs of nitroxyl as inhibitors of aldehyde dehydrogenase.J. Med. Chem. 1992; 35: 3648-3652Crossref PubMed Scopus (61) Google Scholar, 18Nagasawa H.T. DeMaster E.G. Redfern B. Shirota F.N. Goon D.J. Evidence for nitroxyl in the catalase-mediated bioactivation of the alcohol deterrent agent cyanamide.J. Med. Chem. 1990; 33: 3120-3122Crossref PubMed Scopus (117) Google Scholar, 19DeMaster E.G. Redfern B. Nagasawa H.T. Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide.Biochem. Pharmacol. 1998; 55: 2007-2015Crossref PubMed Scopus (154) Google Scholar); and pretreatment of ischemic (oxygen-depleted) tissues with HNO has been shown to protect against ischemia-reperfusion toxicity (20Pagliaro P. Mancardi D. Rastaldo R. Penna C. Gattullo D. Miranda K.M. Feelisch M. Wink D.A. Kass D.A. Paolocci N. Nitroxyl affords thiol-sensitive myocardial protective effects akin to early preconditioning.Free Radic. Biol. Med. 2003; 34: 33-43Crossref PubMed Scopus (181) Google Scholar). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the carbohydrate metabolism pathway, has also been shown to be potently inhibited by HNO both in vitro (19DeMaster E.G. Redfern B. Nagasawa H.T. Mechanisms of inhibition of aldehyde dehydrogenase by nitroxyl, the active metabolite of the alcohol deterrent agent cyanamide.Biochem. Pharmacol. 1998; 55: 2007-2015Crossref PubMed Scopus (154) Google Scholar) and in vivo (21Lopez B.E. Rodriguez C.E. Pribadi M. Cook N.M. Shinyashiki M. Fukuto J.M. Inhibition of yeast glycolysis by nitroxyl (HNO): mechanism of HNO toxicity and implications to HNO biology.Arch. Biochem. Biophys. 2005; 442: 140-148Crossref PubMed Scopus (59) Google Scholar), an effect thought to occur through the direct modification of its active site cysteine. At high concentrations (2–5 mm), HNO has been shown to be cytotoxic by eliciting DNA strand breaks and glutathione depletion, causing cellular toxicity due to oxidative protein damage (22Wink D.A. Feelisch M. Fukuto J. Chistodoulou D. Jourd'heuil D. Grisham M.B. Vodovotz Y. Cook J.A. Krishna M. DeGraff W.G. Kim S. Gamson J. Mitchell J.B. The cytotoxicity of nitroxyl: possible implications for the pathophysiological role of NO.Arch. Biochem. Biophys. 1998; 351: 66-74Crossref PubMed Scopus (185) Google Scholar). However, this toxicological effect is only relevant if physiological HNO levels are high, and it has thus far not been demonstrated to have any in vivo relevance (23Fukuto J.M. Bartberger M.D. Dutton A.S. Paolocci N. Wink D.A. Houk K.N. The physiological chemistry and biological activity of nitroxyl (HNO): the neglected, misunderstood, and enigmatic nitrogen oxide.Chem. Res. Toxicol. 2005; 18: 790-801Crossref PubMed Scopus (155) Google Scholar). These HNO-mediated pharmacological effects are dramatically different from those of NO (11Paolocci N. Katori T. Champion H.C. St John M.E. Miranda K.M. Fukuto J.M. Wink D.A. Kass D.A. Positive inotropic and lusitropic effects of HNO/NO− in failing hearts: independence from β-adrenergic signaling..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5537-5542Crossref PubMed Scopus (282) Google Scholar) most likely because HNO tends to be much more thiophilic with cysteines being the major site of biochemical reactivity (24Fukuto J.M. Dutton A.S. Houk K.N. The chemistry and biology of nitroxyl (HNO): a chemically unique species with novel and important biological activity.Chembiochem. 2005; 6: 612-619Crossref PubMed Scopus (84) Google Scholar, 25Wong P.S. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (329) Google Scholar, 26Paolocci N. Jackson M.I. Lopez B.E. Miranda K. Tocchetti C.G. Wink D.A. Hobbs A.J. Fukuto J.M. The pharmacology of nitroxyl (HNO) and its therapeutic potential: not just the Janus face of NO.Pharmacol. Ther. 2007; 113: 442-458Crossref PubMed Scopus (201) Google Scholar). Therefore, it is no surprise that NO and HNO tend to have different targets. For example, in the vascular system, HNO can act through a cAMP signal transduction pathway, whereas the vascular activity of NO is primarily due to an elevation in cGMP (27Miranda K.M. Paolocci N. Katori T. Thomas D.D. Ford E. Bartberger M.D. Espey M.G. Kass D.A. Feelisch M. Fukuto J.M. Wink D.A. A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9196-9201Crossref PubMed Scopus (248) Google Scholar). Although a number of pharmacological and toxicological effects have been shown for HNO, the underlying mechanisms of action are largely unknown. HNO and cysteine are known to react to produce non-cross-linked sulfinamides and cause disulfide formation, and HNO can react with metals/metalloproteins and oxygen and participate in reduction/oxidation reactions (23Fukuto J.M. Bartberger M.D. Dutton A.S. Paolocci N. Wink D.A. Houk K.N. The physiological chemistry and biological activity of nitroxyl (HNO): the neglected, misunderstood, and enigmatic nitrogen oxide.Chem. Res. Toxicol. 2005; 18: 790-801Crossref PubMed Scopus (155) Google Scholar, 25Wong P.S. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (329) Google Scholar, 26Paolocci N. Jackson M.I. Lopez B.E. Miranda K. Tocchetti C.G. Wink D.A. Hobbs A.J. Fukuto J.M. The pharmacology of nitroxyl (HNO) and its therapeutic potential: not just the Janus face of NO.Pharmacol. Ther. 2007; 113: 442-458Crossref PubMed Scopus (201) Google Scholar). However, the molecular targets of HNO have yet to be linked to its pharmacological and toxicological effects. Here we describe a mass spectrometry-based method for the analysis of the major type of biologically relevant HNO reaction, the reaction with the thiol on cysteines to produce non-cross-linked sulfinamides as well as disulfide linkages (23Fukuto J.M. Bartberger M.D. Dutton A.S. Paolocci N. Wink D.A. Houk K.N. The physiological chemistry and biological activity of nitroxyl (HNO): the neglected, misunderstood, and enigmatic nitrogen oxide.Chem. Res. Toxicol. 2005; 18: 790-801Crossref PubMed Scopus (155) Google Scholar, 25Wong P.S. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (329) Google Scholar, 26Paolocci N. Jackson M.I. Lopez B.E. Miranda K. Tocchetti C.G. Wink D.A. Hobbs A.J. Fukuto J.M. The pharmacology of nitroxyl (HNO) and its therapeutic potential: not just the Janus face of NO.Pharmacol. Ther. 2007; 113: 442-458Crossref PubMed Scopus (201) Google Scholar). Although disulfides are produced through many different pathways, non-cross-linked sulfinamides are exclusively produced by HNO and can thus be used to analyze for the presence of HNO and its effects on cysteine-containing proteins. As well, the sulfinamide modification imparts a specific mass change to cysteines making sulfinamide analysis, and indirectly HNO analysis, very amenable to investigation by mass spectrometry. The sulfinamide modification has been observed in MS spectra (28Hedberg J.J. Griffiths W.J. Nilsson S.J. Hoog J.O. Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry.Eur. J. Biochem. 2003; 270: 1249-1256Crossref PubMed Scopus (64) Google Scholar), and in a recent mass spectrometric analysis, Shen and English (29Shen B. English A.M. Mass spectrometric analysis of nitroxyl-mediated protein modification: comparison of products formed with free and protein-based cysteines.Biochemistry. 2005; 44: 14030-14044Crossref PubMed Scopus (61) Google Scholar) attributed a mass shift of 65 Da on prominent y-ions upon low energy CID to the elimination of the sulfinamide moiety from the molecule in their mass spectrometric comparison of nitroxyl products formed with free and protein-based cysteines. Here we investigate this mass shift and the formation of a previously unstudied neutral loss to determine an efficient method for the identification of the sulfinamide modification and demonstrate its utility on a sample generated by treatment of live platelets immediately post-isolation, that is ex vivo, with HNO. The peptide MHRQETVDCLK-NH2 was provided as a gift from Phil Owen of the Biomedical Research Centre, and peptide EKPLQNFTLCFR-NH2 was purchased from Bachem (Bubendorf, Switzerland). AS (Na2[ONNO2]) was purchased from Cayman Chemicals (Ann Arbor, MI), and sequencing grade trypsin, formic acid, and acetonitrile were purchased from Fisher Scientific. All other reagents were purchased from Sigma-Aldrich. Analysis of the HNO-modified peptides was performed using the following instruments: a nano-ESI (nESI) source triple quadrupole instrument where the third quadrupole (Q3) has linear ion trapping capabilities (2000 Q TRAP), an nESI quadrupole time-of-flight (Q STAR XL) mass spectrometer, and a MALDI-TOF/TOF 4700 Proteomics Analyzer (all Applied Biosystems/MDS Sciex, Concord, Ontario, Canada). Alternatively an nESI linear ion trap coupled to a Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT-ICR) with electron capture dissociation (ECD) capabilities and an nESI Orbitrap instrument were used (Thermo Electron Corp., Bremen, Germany). All analyses were performed with 1–5 μm solutions of the modified peptides. A stock solution of 100 mm AS was prepared in 10 mm NaOH. Nitroxylation was performed on 10 nmol of either MHRQETVDCLK-NH2 or EKPLQNFTLCFR-NH2 with 1 μl of AS stock solution and 99 μl of 15 mm Tris-HCl (pH 7.4) buffer, producing a final AS concentration of 1 mm. Reactions were carried out for 25 min at room temperature. The peptides were then separated from the reactants, purified with C18 ZipTips (Millipore, Billerica, MA), and reconstituted in 50%acetonitrile, 5%formic acid when analyzed by electrospray mass spectrometry or were spotted onto a MALDI target using the dried droplet method and α-cyano-4-hydroxycinnamic acid matrix. The kinetics of the reaction of peptides MHRQETVDCLK-NH2 and EKPLQNFTLCFR-NH2 with HNO was investigated on a Q STAR XL using an ion accumulation time of 1 s, leading to the acquisition of one kinetics measurement per second. HNO is produced from the decomposition of AS upon its addition to the buffered peptide reaction solution with a half-life of 4.4 min at pH 7.4 and 22 °C (30Miranda K.M. Dutton A.S. Ridnour L.A. Foreman C.A. Ford E. Paolocci N. Katori T. Tocchetti C.G. Mancardi D. Thomas D.D. Espey M.G. Houk K.N. Fukuto J.M. Wink D.A. Mechanism of aerobic decomposition of Angeli's salt (sodium trioxodinitrate) at physiological pH.J. Am. Chem. Soc. 2005; 127: 722-731Crossref PubMed Scopus (93) Google Scholar, 31Fukuto J.M. Hobbs A.J. Ignarro L.J. Conversion of nitroxyl (HNO) to nitric oxide (NO) in biological systems: the role of physiological oxidants and relevance to the biological activity of HNO.Biochem. Biophys. Res. Commun. 1993; 196: 707-713Crossref PubMed Scopus (135) Google Scholar). For all kinetics experiments, a 40:1 molar ratio of AS to peptide was used with a final AS concentration of 2 mm. As there was a 40× excess of AS to peptide in the reaction mixture, calculations using the first order rate of HNO decay while ignoring HNO consumption by reaction with thiols or by dehydrative dimerization of HNO showed that HNO may have been in excess compared with the peptide concentration as early as 10 s after AS addition. Two different setups were required to monitor the kinetics of the reaction from 1.7 s to 15 min. For the 1–15-min time points, the reaction solution was prepared by adding AS to 2.5 nmol of either MHRQETVDCLK-NH2 or EKPLQNFTLCFR-NH2 in 12.5 mm Tris-HCl (pH 7.4), 20%methanol; inserted into a nanospray emitter; and then analyzed on the mass spectrometer. For such a process, it took between 1 and 1.08 min from AS mixing into the reaction solution to data collection. Three data sets were collected and averaged, and a five-point moving average was applied to the data. For the shorter time points, kinetics data in the range of 1.7- 90 s were collected based on a continuous flow setup as described previously by Konermann et al. (32Konermann L. Collings B.A. Douglas D.J. Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry.Biochemistry. 1997; 36: 5554-5559Crossref PubMed Scopus (129) Google Scholar) with slight modifications. Briefly two syringes were operated simultaneously with syringe 1 (flow rate of 32 μl/min) containing the buffered peptide mixture and an internal standard (osteocalcin fragment 7–19) and syringe 2 (flow rate of 1 μl/min) containing the AS solution. Each syringe was connected to a fused silica capillary (100-μm inner diameter, Polymicro Technologies, Phoenix, AZ) that was then connected together by a splitter on the electrospray source (MDS Sciex) to a third reaction capillary (also 100-μm inner diameter fused silica) that led the sample to the tip of the emitter. The length of the third capillary was varied from 627 to 36 cm corresponding to reaction times from 89.5 to 5.1 s (32Konermann L. Collings B.A. Douglas D.J. Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry.Biochemistry. 1997; 36: 5554-5559Crossref PubMed Scopus (129) Google Scholar). To reach reaction times as low as 1.7 s, the flow rate of the two syringes was increased proportionately to obtain total flow rates up to 100 μl/min. An accumulation time of 1 s per mass spectrum with a total acquisition time of 1 min was collected for one data set. The internal standard, osteocalcin fragment 7–19, was used to normalize the intensities of the precursor, intermediates, and final product as they may vary with the flow rate. Ethical approval for this study was granted by the University of British Columbia Research Ethics Board, and informed consent was granted by the donors. Whole blood was drawn from the antecubital vein of healthy human volunteers into 0.15%(v/v) acid-citrate-dextrose anticoagulant. Platelets were isolated by centrifugation, washed twice in physiological buffer (10 mm trisodium citrate, 30 mm dextrose, 1 IU/ml apyrase), and resuspended at physiological concentration (200–350 × 109/liter) in Tris-buffered saline containing 5 mm EDTA. Platelets were treated with either AS (10 μm, 100 μm, 1 mm, and 10 mm) from a stock of 1 m AS in 10 mm NaOH or a vehicle control for 2 min at room temperature. Samples were spun at 1000 × g for 3 min at room temperature to pellet the platelets. Platelet pellets were resuspended in 1× lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1%Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm Na3VO4, 1× protease inhibitor mixture), and lysates were immediately snap frozen in liquid N2. Samples were thawed slowly on ice, mixed with non-reducing sample buffer, and separated by one-dimensional (1D) SDS-PAGE. Gels were stained with Coomassie Brilliant Blue, and bands of interest were excised. Tryptic in-gel digestions were performed overnight at 37 °C without reducing or alkylating the sample (33Park Z.Y. Russell D.H. Thermal denaturation: a useful technique in peptide mass mapping.Anal. Chem. 2000; 72: 2667-2670Crossref PubMed Scopus (152) Google Scholar). Following digestion, peptides were extracted for MS analysis. Identification of peptides with HNO-induced modifications from the in-gel digests of samples treated with 10 mm AS was performed by nano-HPLC MS/MS on an Agilent 1100 instrument (Agilent, Santa Clara, CA) coupled to an LTQ-Orbitrap using a 15-cm-long, 75-μm-inner diameter fused silica column packed with 3-μm-particle size reverse phase (C18) beads (Dr. Maisch GmbH) using water:acetonitrile:formic acid as the mobile phase with gradient elution. Identification of modified peptides was performed by extracting the Mascot generic format files from the MS data using DTA Super Charge, part of the MSQuant open source project (SourceForge, Inc.) and then searching them against the human Swiss-Prot database (v.54.5; 589,473 sequences) using Mascot v.2.1 (34Science Matrix Mascot. 2007; (Matrix Science, Boston)Google Scholar). The following search criteria were used: trypsin cleavage specificity with up to one missed cleavage site, no fixed modifications, variable modifications of oxidized methionine and dioxidized cysteine (sulfinic acid), ±10-ppm peptide tolerance, ±0.6-Da MS/MS tolerance, and the scoring scheme was ESI-TRAP. All MS/MS spectra of peptides that were identified as containing sulfinic acid modifications by Mascot were inspected manually to confirm the peptide assignment. These candidate modified peptides were then selected for multiple reaction monitoring (MRM) analysis for the dose-response experiment with the product ion spectra from the LTQ of the Orbitrap being used to determine the MRM transitions. The dose-response analysis was completed with MRM experiments performed by nano-HPLC MS/MS on an Ultimate pump (LC Packings, Sunnyvale, CA) using a 15-cm-long, 75-μm-inner diameter, 3-μm-particle size reverse phase (C18) column (LC Packings) coupled to an ESI 2000 Q TRAP. Each putative modified protein identified by the Orbitrap was then monitored by four MRM transitions (Table I): one to monitor the precursor ion with no fragmentation in the collision cell and three MRM transitions to monitor three different fragment ions produced from fragmentation of the precursor in the collision cell. Peak areas from these three fragment ions were collected from the extracted ion chromatograms and then averaged to increase selectivity as well as to allow for relative quantitation of the amount of sulfinic acid modification at different doses.Table IModified peptides identified by the Orbitrap and the associated MRM transitions for the dose-response experimentProteinPeptide(s)MRM transitionNeutral lossModified precursorInternal standardsTalinVVAPTISSPVCQEQLVEAGR (722–741)1058.04 → 1058.04415.26 → 415.26Yes (66 Da)1058.04 → 1360.65415.26 → 629.401058.04 → 1029.531017.51 → 1017.511058.04 → 432.221017.51 → 1405.67Glycoprotein 1b" @default.
• W1997455293 created "2016-06-24" @default.
• W1997455293 creator A5037342475 @default.
• W1997455293 creator A5053411192 @default.
• W1997455293 creator A5067053556 @default.
• W1997455293 creator A5072948450 @default.
• W1997455293 date "2009-05-01" @default.
• W1997455293 modified "2023-10-18" @default.
• W1997455293 title "Identification of Nitroxyl-induced Modifications in Human Platelet Proteins Using a Novel Mass Spectrometric Detection Method" @default.
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