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A tandem mass spectrometry study of the transnitrosylation products of the dipeptide cysteinyl-tryptophan Introduction Mass spectrometry provides a suitable technique for the study of the formation and fragmentation of peptide radical ions. Thiyl radicals occur in biological processes such as enzyme catalysis and oxidative stress. Generation of thiyl and indolyl radical cations can be accomplished in the gas-phase by cleavage of the labile S?NO and N-NO bonds in the protonated species of S-nitrosopeptides and N-nitrosopeptides, respectively.Employing this strategy, the structures, reactivities and fragmentation of a series of radical cations obtained from cysteine- and tryptophan- containing oligopeptides have been studied [1,2]. In a previous study [3], protonated dinitrosylated cysteinyl-cysteine was obtained in the gas phase affording a protonated cyclic structure with a disulfide bridge upon the loss of 2 NO molecules. MethodsSynthetic peptide cysteinyl-tryptophan (Cys-Trp, CW) was prepared by means of solid-phase peptide synthesis. Transnitrosylation reactions in solution were carried out mixing a stock solution of the dipeptide Cys-Trp with tert-butyl nitrite (TBN). The reaction mixture was left 10 minutes at room temperature with occasional stirring before dillution and infusion into the ESI source. Tandem mass spectrometry experiments were carried out using a hybrid triple quadrupole/ linear ion-trap mass spectrometer or a quadrupole ion-trap. High resolution mass spectra were acquired using a hybrid linear ion-trap-Orbitrap mass spectrometer. Product ions were generated (MS/MS) from the protonated peptides and then their fragmentation behaviors (MS/MS/MS) were analyzed. Quantum chemical calculations were carried out at the UB3LYP/6-311++G(d,p) level of theory on the ions.Preliminary dataThe nitrosylation reaction between TBN and Cys-Trp can modify both the thiol and the indole nitrogen groups in the dipeptide giving rise to mono- or di-nitrosylated products. Protonated mononitrosylated dipeptide upon loss of the NO forms a radical cation, which in turn shows three major fragmentations: losses of CO2, cysteinamide and a radical loss that creates the 3-methylene indole cation.Protonated dinitrosylated dipeptide dissociates by losing one NO radical first and then the other, generating an ion that is potentially a diradical and is denoted as [ox-CW+H]+. The major dissociation channel of this product ion was loss of ammonia although a variety of other neutral molecule losses were also observed. The fragmentation pathways of the protonated dimer of the dipeptide containing cystine, [(CW)2 + H]+ were studied looking for further evidence to clarify the structure of the product ion obtained upon loss of the 2 NO groups. Cleavage of the S-S bond of [(CW)2 + H]+ gave a product ion at m/z 306 that is isobaric with [ox-CW+H]+. These two m/z 306 ions were found to have identical CID spectra.DFT calculations do not show significant differences in the energies involved for the loss of the NO radical from either the cysteine or the tryptophan residues of the protonated di-nitrosylated dipeptide. However, upon loss of the NO radical from the tryptophan, a delocalized -radical cation on the indole ring is formed. This stabilized structure may displace the remaining NO from the S by radical substitution, generating a cyclic structure, as was observed in the dissociation of [CySNO-CySNO+H]+ [3]. [1] Knudsen E.R., Julian R.R. Int. J. Mass Spectrom. 2010, 294, 83-87.[2] Osburn S., Berden G., Oomens J., O´Hair R.A.J., Ryzhov V. J.Am. Soc.Mass Spectrom. 2012, 23, 1019-1023.[3] Butler M., Siu K.W.M, Hopkinson A.C. Phys. Chem. Chem. Phys. 2016, DOI: 10.1039/c5cp08014bNovel aspectThe gas-phase chemistry posed after the loss of two NO moieties from the protonated dinitrosylated dipeptide cysteinyl-tryptophan is examined

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