Gas-phase fragmentation of long-lived cysteine radical cations formed via no loss from protonated S-nitrosocysteine

Abstract

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 •ϩ ), which fragments under collision-induced dissociation (CID) conditions via losses in the following relative abundance order: •COOH ӷ CH 2 S Ͼ •CH 2 SH Ϸ H 2 S. 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•ϩ with a sulfur radical site undergoes proton and/or H atom transfer reactions that precede the losses of T here has been renewed interest in the gas-phase formation and reactions of radical ions of biomolecules. The motivation for these studies range from fundamental interest in species related to biological processes such as enzyme catalysis [1] and oxidative chemistry associated with damage to biomolecules such as DNA [2] and proteins [3] through to the potential for developing novel mass spectrometry based analytical applications [4]. With regards to the formation of radical ions of amino acids and peptides, several methods have been developed as alternatives to electron ionization (EI) [5]. These include UV photodissociation [6 -11] and ion-electron based techniques such as electron capture dissociation (ECD) [12][13][14][15]. Chemical-based methods that involve low-energy collisioninduced dissociation (CID) [16] of ions generated via electrospray ionization and which can thus be carried out on a wide range of mass spectrometers, merit special discussion. The first, pioneered by Siu, involves carrying out CID of ternary metal complexes to form radical cations of peptides via the redox reaction shown in eq 1 [17][18][19]. To date, doubly charged copper(II) ternary metal complexes (eq 1, where Metal ϭ Cu; L ϭ a range of neutral ligands, x ϭ 2, y ϭ 0) and singly charged metal(III) complexes (eq 1, where Metal ϭ Cr, Mn, Fe, and Co; L ϭ a salen ligand, x ϭ 3, y ϭ 2) have been used to study the formation and reactions of peptide radical ions, including recent contributions from Julia Laskin [20 -23].The second method involves carrying out CID on a cationized peptide containing a functional group with a weak bond, which is susceptible to bond homolysis. A number of different functional groups have been examined to date including: (1) N-terminal azo derivatives

The Radical Ion Chemistry of S-Nitrosylated Peptides

“…The S-nitrosopeptides were also subjected to CID, and in agreement with previous findings [5,22], the dominant fragmentation channel was loss of • NO. The long-lived radical peptide ion was subjected to ECD, providing an interesting comparison with ECD of the unmodified (hydrogen abundant) peptide.…”

“…These losses were also observed in the MS 3 experiments of Hao and Gross [5], but were not observed following fragmentation of cysteine radical cations [22]. In the latter case, losses of H 2 S were observed.…”

“…On formation, the radical resides on the sulfur atom (the distonic structure). Both Zhao et al [36] and Ryzhov et al [22], calculated that the most stable structure was the result of hydrogen atom transfer from the α-carbon giving an α-radical stabilized by the captodative effect [37]. Nevertheless, gas-phase IR spectroscopy by Fornarini and co-workers revealed the long-lived distonic structure of the radical [28].…”

“…• NO neutral loss is observed following CID of Snitrosopeptides [5,22,[26][27][28], which might suggest a thermal process is occurring; however, no loss of + ions were observed when the energy of the electrons was reduced (Supplemental Figure 3).…”

Photodissociation of TEMPO-modified peptides: new approaches to radical-directed dissociation of biomolecules

“…Subsequent activation of these radical ions initiates radical-driven fragmentation along the peptide backbone and thus yields a range of diagnostic product ions. [20][21][22][23][24] Chemical modification of the N-terminus of the peptide or protein is another example of derivitisation for FRIPS. For instance, upon CID, peroxycarbamates 25 and azo moieties 26 are susceptible to homolytic dissociation forming nitrogen-and carbon-centred radicals, respectively.…”