Victor Ryzhov

Gas-Phase Fragmentation of Long-Lived Cysteine Radical Cations Formed Via NO Loss from Protonated S-Nitrosocysteine

In this work, we describe two different methods for generating protonated S-nitrosocysteine in the gas phase. The first method involves a gas-phase reaction of protonated cysteine with t-butylnitrite, while the second method uses a solution-based transnitrosylation reaction of cysteine with S-nitrosoglutathione followed by transfer of the resulting S-nitrosocysteine into the gas phase by electrospray ionization mass spectrometry (ESI-MS). Independent of the way it was formed, protonated S-nitrosocysteine readily fragments via bond homolysis to form a long-lived radical cation of cysteine (Cys•+), which fragments under collision-induced dissociation (CID) conditions via losses in the following relative abundance order: •COOH ≫ CH2S > •CH2SH-H2S. Deuterium labeling experiments were performed to study the mechanisms leading to these pathways. DFT calculations were also used to probe aspects of the fragmentation of protonated S-nitrosocysteine and the radical cation of cysteine. NO loss is found to be the lowest energy channel for the former ion, while the initially formed distonic Cys•+ with a sulfur radical site undergoes proton and/or H atom transfer reactions that precede the losses of CH2S, •COOH, •CH2SH, and H2S.

Mobile Protons Versus Mobile Radicals: Gas-Phase Unimolecular Chemistry of Radical Cations of Cysteine-Containing Peptides⁎†

A combination of electrospray ionization (ESI), multistage, and high-resolution mass spectrometry experiments are used to examine the gas-phase fragmentation reactions of radical cations of cysteine containing di- and tripeptides. Two different chemical methods were used to form initial populations of radical cations in which the radical sites were located at different positions: (1) sulfur-centered cysteinyl radicals via bond homolysis of protonated S-nitrosocysteine containing peptides; and (2) α-carbon backbone-centered radicals via Siu’s sequence of reactions (J. Am. Chem. Soc.2008, 130, 7862). Comparison of the fragmentation reactions of these regiospecifically generated radicals suggests that hydrogen atom transfer (HAT) between the α C-H of adjacent residues and the cysteinyl radical can occur. In addition, using accurate mass measurements, deuterium labeling, and comparison with an authentic sample, a novel loss of part of the N-terminal cysteine residue was shown to give rise to the protonated, truncated N-formyl peptide (an even-electron xn ion). DFT calculations were performed on the radical cation [GCG].+ to examine: the relative stabilities of isomers with different radical and protonation sites; the barriers associated with radical migration between four possible radical sites, [G.CG]+, [GC.G]+, [GCG.]+, and [GC(S.)G]+; and for dissociation from these sites to yield b2-type ions.

Structure and Reactivity of the Cysteine Methyl Ester Radical Cation

The structure and reactivity of the cysteine methyl ester radical cation, CysOMe(.+) , have been examined in the gas phase using a combination of experiment and density functional theory (DFT) calculations. CysOMe(.+) undergoes rapid ion-molecule reactions with dimethyl disulfide, allyl bromide, and allyl iodide, but is unreactive towards allyl chloride. These reactions proceed by radical atom or group transfer and are consistent with CysOMe(.+) possessing structure 1, in which the radical site is located on the sulfur atom and the amino group is protonated. This contrasts with DFT calculations that predict a captodative structure 2, in which the radical site is positioned on the α carbon and the carbonyl group is protonated, and that is more stable than 1 by 13.0 kJ mol(-1) . To resolve this apparent discrepancy the gas-phase IR spectrum of CysOMe(.+) was experimentally determined and compared with the theoretically predicted IR spectra of a range of isomers. An excellent match was obtained for 1. DFT calculations highlight that although 1 is thermodynamically less stable than 2, it is kinetically stable with respect to rearrangement.

Coupling of ion-molecule reactions with liquid chromatography on a quadrupole ion trap mass spectrometer

We report for the first time a coupling of gas-phase ion-molecule reactions with chromatographic separations on a quadrupole ion trap mass spectrometer. The interface was accomplished by using a pulsed valve for the introduction of a volatile neutral into the ion trap. The pulsed valve controller is synchronized with the mass spectrometer software. The setup requires some minor modifications to the vacuum system of the commercial quadrupole ion trap but most of the modifications are external to the mass spectrometer. Two applications of this interface are described: differentiation between two phosphoglucose positional isomers and detection of a phosphopeptide in a peptide mixture. Both applications are using the reactivity of trimethoxyborate towards a phosphate moiety in the negative ion mode. The detection of phosphopeptides hinges on our findings that non-phosphorylated peptide anions do not react with trimethoxyborate. This LC/MS detection can be easily visualized in terms of selected reaction monitoring.

Binding of metalloporphyrins to model nitrogen bases: collision-induced dissociation and ion–molecule reaction studies

Abstract Binding of several volatile nitrogen bases to four metalloporphyrin cations was studied by collision-induced dissociation (CID) as well as ion–molecule reactions in a quadrupole ion trap mass spectrometer. Relative binding energy order was obtained from the CID data by comparing the stability of metalloporphyrin–amine complexes while accounting for the effects of the complex size. The efficiencies of ion–molecule association reactions were also used to compare binding in these systems. The results from these two approaches agree with each other. There appears to be no direct correlation between the proton affinity of model bases with their metalloporphyrin affinity. This discrepancy is rationalized in terms of steric hindrance and dipole moment effects.

Structure and Reactivity of Homocysteine Radical Cation in the Gas Phase Studied by Ion–Molecule Reactions and Infrared Multiple Photon Dissociation

The reactivity of the cysteine (Cys) and homocysteine (Hcy) radical cation was studied using ion-molecule reactions. The radical cations were generated via collision-induced dissociation (CID) of their S-nitrosylated precursors. Cleavage of the S-NO bond led to the formation of the radical initially positioned on the sulfur atom. The reactions of the radical cations with dimethyl disulfide revealed that the cysteine radical cation reacts more quickly than the homocysteine radical cation. Infrared multiple photon dissociation (IRMPD) spectroscopy and density functional theory (DFT) calculations were used to determine the structure of the homocysteine radical cation, which was compared to the previously published structure of the cysteine radical cation (Sinha et al. Phys. Chem. Chem. Phys. 2010, 12, 9794-9800). IRMPD spectroscopy and DFT calculations revealed that this difference in radical reactivity was not a result of a radical rearrangement for the homocysteine radical cation but rather that the reactivity was modulated by stronger hydrogen bonding.

Mass Spectroscopy and Molecular Modeling Predict Endothelial Nitric Oxide Synthase Dimer Collapse by Hydrogen Peroxide Through Zinc Tetrathiolate Metal-Binding Site Disruption

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

Comparison of collision- versus electron-induced dissociation of Pt(II) ternary complexes of histidine- and methionine-containing peptides.

Incubation of the histidine-containing peptides (GH, HG, GGH, GHG, HGG) and methionine-containing peptides (GM, MG, GGM, GMG, MGG) with the platinum complexes [Pt(terpy)Cl](+) (A) and [Pt(dien)Cl](+) (B) followed by electrospray ionisation (ESI) led to a number of singly and doubly charged ternary platinum peptide complexes, including [Pt(L)M](2+) and [Pt(L)M-H](+) (where L = the ligand terpy or dien; M is a peptide). Each of the [Pt(L)M](2+) complexes was subjected to electron capture dissociation (ECD), collision-induced dissociation (CID) and electron-induced dissociation (EID), while each of the [Pt(L)M-H](+) complexes was subjected to CID and EID. Results from ECD suggest that the free electron is captured by the metal ion thus weakening the bonds to its ligands. In the case of the ligand terpy, which binds more strongly than dien, this weakening leads to the loss of the peptide. The minor products in the ECD spectra of [Pt(terpy)M](2+) complexes do show fragmentation along the peptide backbone, but the ions observed are of the a-, b-, and y-type. For the complexes with methionine-containing peptides, a marker ion, [Pt(L)SCH(3)](+), was found which is indicative of binding of Pt to the methionine side chain. For the histidine-containing peptides, an ion containing platinum, the auxiliary ligand, and the histidine imine was observed in many instances, thus indicating the binding of the histidine side chain to the metal, but other modes of Pt coordination (N-terminus) were also found to be competitive. These findings are consistent with a recent finding (Sze et al. J. Biol. Inorg. Chem. 2009; 14: 163) that Pt occupies the methionine-rich copper(I)-binding site rather than histidine-rich copper(II)-binding site in the CopC protein.

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post-translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.

Post-translational modification in the gas phase: mechanism of cysteine S-nitrosylation via ion-molecule reactions.

The gas-phase mechanism of S-nitrosylation of thiols was studied in a quadrupole ion trap mass spectrometer. This was done via ion-molecule reactions of protonated cysteine and many of its derivatives and other thiol ions with neutral tert-butyl nitrite or nitrous acid. Our results showed that the presence of the carboxylic acid functional group, -COOH, in the vicinity of the thiol group is essential for the gas-phase nitrosylation of thiols. When the carboxyl proton is replaced by a methyl group (cysteine methyl ester) no nitrosylation was observed. Other thiols lacking a carboxylic acid functional group displayed no S-nitrosylation, strongly suggesting that the carboxyl hydrogen plays a key role in the nitrosylation process. These results are in excellent agreement with a solution-phase mechanism proposed by Stamler et al. (J. S. Stamler, E. J. Toone, S. A. Lipton, N. J. Sucher. Neuron 1997, 18, 691-696) who suggested a catalytic role for the carboxylic acid group adjacent to cysteine residues and with later additions by Ascenzi et al. (P. Ascenzi, M. Colasanti, T. Persichini, M. Muolo, F. Polticelli, G. Venturini, D. Bordo, M. Bolognesi. Biol. Chem. 2000, 381, 623-627) who postulated that the presence of the carboxyl in the cysteine microenvironment in proteins is crucial for S-nitrosylation. A concerted mechanism for the gas-phase S-nitrosylation was proposed based on our results and was further studied using theoretical calculations. Our calculations showed that this proposed pathway is exothermic by 44.0 kJ mol(-1). This is one of the few recent examples when a gas-phase mechanism matches one in solution.