Evidence for the Prerequisite Formation of Phenoxyl Radicals in Radical-Mediated Peptide Tyrosine Nitration In Vacuo.

The elementary mechanism of radical-mediated peptide tyrosine nitration, which is a hallmark of post-translational modification of proteins under nitrative stress in vivo, has been elucidated in detail by using an integrated approach that combines the gas-phase synthesis of prototypical molecular tyrosine-containing peptide radical cations, ion-molecule reactions, and isotopic labeling experiments with DFT calculations. This reaction first involves the radical recombination of . NO2 towards the prerequisite phenoxyl radical tautomer of a tyrosine residue, followed by proton rearrangements, finally yielding the stable and regioselective 3-nitrotyrosyl residue product. In contrast, nitration with the π-phenolic radical cation tautomer is inefficient. This first direct experimental evidence for the elementary steps of the radical-mediated tyrosine nitration mechanism in the gas phase provides a fundamental insight into the regioselectivity of biological tyrosine ortho-nitration.

Doubly Charged Small Organic Fragments Derived from [Ce(tripeptide)(CH3CN)m]3+ Complexes: Observation of the Elusive [bn + H]2+ Ions.

[a3 + H]2+ ions generated from Ln3+/tripeptide complexes, where Ln = La or Ce, have similar structures to the linear [an]+ ions but with protonation at both the terminal NH2 and N═CH2 groups. Ion stability is favored by having the basic secondary amine of the proline residue at the N-terminus and by an amino acid residue accommodating one of the protons on the side chain. Dissociation of [a3 + H]2+ ions derived from peptides containing only aliphatic residues is by cleavage of the second amide bond to give [b2]+ or [a2]+ ions along with internal [a1]+ ions. For [a3 + H]2+ ions containing a tryptophan residue in the central location, in addition to cleavage of the amide bond, losses of neutrals NH3, HN═CHR, (NH3 + CO), and HNCO were observed. Dissociations of some unsolvated Ln3+/tripeptide complexes gave [b3 + H]2+ ions in low abundance; formation of these [b3 + H]2+ ions was favored by the presence of a proline residue at the N-terminus and by either a histidine or tryptophan residue in the central position. Dissociation of these [b3 + H]2+ ions was by the loss of (H2O + CO) and not only CO, indicating that these ions did not have the same type of oxazolone structure as found for [bn]+ ions. Density functional theory calculations suggest that the observed [b3 + H]2+ ions of ProGlyGly were formed from [Ce(ProGlyGly)]3+ complexes in which the peptide was bound to the metal ion as an enolate. Dissociation of the slightly lower-energy complex, where the peptide is bound in the keto form, would produce an oxazolone but the high barrier required to create this isomer of the [b3 + H]2+ ion would be sufficient to result in further dissociation. Two isomers of the [b3 + H]2+ ion of ProHisGly have been created, one from the [Ce(ProHisGly)]3+ complex that characteristically dissociates by the combined loss of (H2O + CO) and the other by the loss of glycine from [ProHisGlyGly + 2H]2+. The [b3 + H]2+ ion derived from [ProHisGlyGly + 2H]2+ dissociated by the loss of only CO.

Structures and Dissociation Products of Ce/Peptide Complexes: Competition between Coordination and Charge Delocalization.

Structures of [Ce(GGG)]3+ and [Ce(GGG ? H)]2+ have been investigated by DFT calculations. The two lowest-energy structures of the triply charged metal complex have the peptide in either the iminol or conventional zwitterionic form, and these ions have almost identical energies. In the doubly charged complex, the iminol and charge-solvated structures are the best structures on the potential energy surface, but the latter is favored. In both iminol structures, the metal ion coordinates to the iminol oxygen rather than to the nitrogen, unlike in previously reported iminol-containing complexes. Triply charged [Ce(peptide)]3+ complexes are fragile and not easily isolated in a mass spectrometer, whereas the doubly charged [Ce(peptide ? H)]2+ complexes are more robust. Here, we studied the fragmentations of 37 [Ce(peptide ? H)]2+ and 30 [Ce(peptide)(peptide ? H)]2+ complexes and the results are systematically summarized. Losses of CO and/or H2O are the most commonly observed fragmentation channels for [Ce(peptide ? H)]2+ complexes and these dissociation pathways are modeled by DFT calculations. For [Ce(peptide)(peptide ? H)]2+ complexes the neutral peptide plays the role of a solvent molecule but, unlike in the dissociations of [Ce(CH3CN)(peptide ? H)]2+ complexes, the loss of the solvent molecule is not observed. Instead, fragmentation occurs by cleavage of the second amide bond of the solvating peptide molecule.

Loss of water from protonated polyglycines: interconversion and dissociation of the product imidazolone ions.

Collision-induced dissociation of isotopically labelled protonated pentaglycine produced two abundant [b5]+ ions, the products of the loss of water from the first and second amide groups, labelled [b5]+I and [b5]+II. IRMPD spectroscopy and DFT calculations show that these two [b5]+ ions feature N1-protonated 3,5-dihydro-4H-imidazol-4-one structures. 15N-Labelling established that some interconversion occurs between these two ions but dissociations are preferred. For both ions, DFT calculations show that the barrier to interconversion is slightly higher than those to dissociation. Dehydration of protonated hexaglycine produced three imidazolone ions. Ions [b6]+I and [b6]+II exhibit analogous CID spectra to those from [b5]+I and [b5]+II; however, the spectrum of the [b6]+III ion was dramatically different, showing losses predominantly of a further water molecule or cleavage of the second amide bond to give the glycyloxazolone (a deprotonated [b2]+ ion, labelled GlyGlyox (114 Da)) from the N-terminus. Protonated polyglycines [Glyn + H]+, where n = 7-9, all readily lose at least one water molecule. The corresponding [bn]+ ions lose either a further water molecule, an oxazolone from the N-terminus or a truncated peptide from the C-terminus. The number of amino acid residues in the latter two eliminated neutral molecules provides insight into the location of the imidazolone in the peptide chain and which oxygen was lost in the initial dehydration reaction. From this analysis, it appears that water loss from the longer protonated polyglycines is predominantly from the central residues.

Interconversion between 4-Imidazolone Ions; Isomers of [b4]+ Derived from Protonated Tetraglycine.

Collision-induced dissociations of isotopically labeled protonated tetraglycines establish that the [b4]+ ion formed by loss of water from the second amide bond (structure II) rearranges to form N1-protonated 3,5-dihydro-4H-imidazol-4-one (structure I), the product of water loss from the first amide bond. Structure II is slightly higher in energy than I (ΔH at 0 K is 5.1 kJ mol-1, as calculated at M06-2X/6-311++G-(d,p)), and the barrier to interconversion is 139.8 kJ mol-1 above I. The dominant dissociation pathway is the loss of methanimine (HN=CH2) from ion I with a barrier of 167.1 kJ mol-1, giving [GlyGlyGlyGly + H - H2O - HN=CH2]+, ion III; a minor channel, loss of NH3, has a slightly higher barrier (181.5 kJ mol-1). Using labeled glycine (13Cα) it was determined that loss of the imine is from the same residue as that from which water was initially lost. The collision-induced dissociation spectra of ion III derived from both I and II were identical, and their energy-resolved curves were also very similar. Ion III fragments by losses of a glycine molecule (the dominant channel), a water molecule, and a glycine residue (57 Da), giving ions IV, V, and VII, respectively. Isotopic labeling established the origins of each of the neutral molecules that are lost. Using glycine (2,2 D2), rapid deuterium exchange was observed for both ions I and II for the α-hydrogens that are from the same residue as that from which the water had been eliminated.

Isomerization versus dissociation of phenylalanylglycyltryptophan radical cations.

Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ˙]+, α-carbon- [FGα˙W]+, N-centered [FGWN˙]+ and ζ-carbon- [Fζ˙GW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal-ligand-peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen-nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα˙W]+, and ζ-phenyl radical [Fζ˙GW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ˙]+ and [FGWN˙]+ is only 31.1 kcal mol-1, much lower than the barriers to their dissociation (40.3 kcal mol-1). For the [FGWπ˙]+, [FGα˙W]+, and [Fζ˙GW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 - H]˙+ ions isolated from [FGα˙W]+ and [Fζ˙GW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.

Ground and Excited-Electronic-State Dissociations of Hydrogen-Rich and Hydrogen-Deficient Tyrosine Peptide Cation Radicals.

We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H](+●) and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS(3) dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%-94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in Cu(II)(2,2':6',2″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR](+●) that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates. Graphical Abstract ᅟ.

Nucleophilic substitution by amide nitrogen in the aromatic rings of [zn - H]˙⁺ ions; the structures of the [b2 - H - 17]˙⁺ and [c1 - 17]⁺ ions.

Peptide radical cations that contain an aromatic amino acid residue cleave to give [zn - H]˙⁺ ions with [b2 - H - 17]˙⁺ and [c1 - 17](+) ions, the dominant products in the dissociation of [zn - H]˙⁺, also present in lower abundance in the CID spectra. Isotopic labeling in the aromatic ring of [Yπ˙GG](+) establishes that in the formation of [b2 - H - 17]˙⁺ ions a hydrogen from the δ-position of the Y residue is lost, indicating that nucleophilic substitution on the aromatic ring has occurred. A preliminary DFT investigation of nine plausible structures for the [c1 - 17](+) ion derived from [Y(π)˙GG](+) shows that two structures resulting from attack on the aromatic ring by oxygen and nitrogen atoms from the peptide backbone have significantly better energies than other isomers. A detailed study of [Y(π)˙GG](+) using two density functionals, B3LYP and M06-2X, with a 6-31++G(d,p) basis set gives a higher barrier for attack on the aromatic ring of the [zn - H]˙⁺ ion by nitrogen than by the carbonyl oxygen. However, subsequent rearrangements involving proton transfers are much higher in energy for the oxygen-substituted isomer leading to the conclusion that the [c1 - 17](+) ions are the products of nucleophilic attack by nitrogen, protonated 2,7-dihydroxyquinoline ions. The [b2 - H - 17]˙⁺ ions are formed by loss of glycine from the same intermediates involved in the formation of the [c1 - 17](+) ions.

Formation, isomerization, and dissociation of ε- and α-carbon-centered tyrosylglycylglycine radical cations.

The fragmentation products of the ε-carbon-centered radical cations [Y(ε)˙LG](+) and [Y(ε)˙GL](+), made by 266 nm laser photolysis of protonated 3-iodotyrosine-containing peptides, are substantially different from those of their π-centered isomers [Y(π)˙LG](+) and [Y(π)˙GL](+), made by dissociative electron transfer from ternary metal-ligand-peptide complexes. For leucine-containing peptides the major pathway for the ε-carbon-centered radical cations is loss of the side chain of the leucine residue forming [YG(α)˙G](+) and [YGG(α)˙](+), whereas for the π-radicals it is the side chain of the tyrosine residue that is lost, giving [G(α)˙LG](+) and [G(α)˙GL](+). The fragmentations of the product ions [YG(α)˙G](+) and [YGG(α)˙](+) are compared with those of the isomeric [Y(ε)˙GG](+) and [Y(π)˙GG](+) ions. The collision-induced spectra of ions [Y(ε)˙GG](+) and [YGG(α)˙](+) are identical, showing that interconversion occurs prior to dissociation. For ions [Y(ε)˙GG](+), [Y(π)˙GG](+) and [YG(α)˙G](+) the dissociation products are all distinctly different, indicating that dissociation occurs more readily than isomerization. Density functional theory calculations at B3LYP/6-31++G(d,p) gave the relative enthalpies (in kcal mol(-1) at 0 K) of the five isomers to be [Y(ε)˙GG](+) 0, [Y(π)˙GG](+) -23.7, [YGG(α)˙](+) -28.7, [YG(α)˙G](+) -31.0 and [Y(α)˙GG](+) -38.5. Migration of an α-C-H atom from the terminal glycine residue to the ε-carbon-centered radical in the tyrosine residue, a 1-11 hydrogen atom shift, has a low barrier, 15.5 kcal mol(-1) above [Y(ε)˙GG](+). By comparison, isomerization of [Y(ε)˙GG](+) to [YG(α)˙G](+) by a 1-8 hydrogen atom migration from the α-C-H atom of the central glycine residue has a much higher barrier (50.6 kcal mol(-1)); similarly conversion of [Y(ε)˙GG](+) into [Y(π)˙GG](+) has a higher energy (24.4 kcal mol(-1)).

Discovery and mechanistic studies of facile N-terminal Cα-C bond cleavages in the dissociation of tyrosine-containing peptide radical cations.

Fascinating N-terminal Cα-C bond cleavages in a series of nonbasic tyrosine-containing peptide radical cations have been observed under low-energy collision-induced dissociation (CID), leading to the generation of rarely observed x-type radical fragments, with significant abundances. CID experiments of the radical cations of the alanyltyrosylglycine tripeptide and its analogues suggested that the N-terminal Cα-C bond cleavage, yielding its [x2 + H](•+) radical cation, does not involve an N-terminal α-carbon-centered radical. Theoretical examination of a prototypical radical cation of the alanyltyrosine dipeptide, using density functional theory calculations, suggested that direct N-terminal Cα-C bond cleavage could produce an ion-molecule complex formed between the incipient a1(+) and x1(•) fragments. Subsequent proton transfer from the iminium nitrogen atom in a1(+) to the acyl carbon atom in x1(•) results in the observable [x1 + H](•+). The barriers against this novel Cα-C bond cleavage and the competitive N-Cα bond cleavage, forming the complementary [c1 + 2H](+)/[z1 - H](•+) ion pair, are similar (ca. 16 kcal mol(-1)). Rice-Ramsperger-Kassel-Marcus modeling revealed that [x1 + H](•+) and [c1 + 2H](+) species are formed with comparable rates, in agreement with energy-resolved CID experiments for [AY](•+).

Laser-induced dissociation of singly protonated peptides at 193 and 266 nm within a hybrid linear ion trap mass spectrometer.

RATIONALE: We implemented, for the first time, laser-induced dissociation (LID) within a modified hybrid linear ion trap mass spectrometer, QTrap, while preserving the original scanning capabilities and routine performance of the instrument. METHODS: Precursor ions of interest were mass-selected in the first quadrupole (Q1), trapped in the radiofrequency-only quadrupole (q2), photodissociated under irradiation with a 193- or 266-nm laser beam in the third quadrupole (q3), and mass-analyzed using the linear ion trap. RESULTS: LID of singly charged protonated peptides revealed, in addition to conventional amide-bond cleavages, preferential fragmentation at Cα -C/N-Cα bonds of the backbone as well as at the Cα -Cß /Cß -Cγ bonds of the side-chains. The LID spectra of [M+H](+) featured product ions that were very similar to the observed radical-induced fragmentations in the CID spectra of analogous odd-electron radical cations generated through dissociative electron-transfer in metal-ligand-peptide complexes or through laser photolysis of iodopeptides. CONCLUSIONS: LID of [M+H](+) ions results in fragmentation channels that are comparable with those observed upon the CID of M(•+) ions, with a range of fascinating radical-induced fragmentations.

Formation and dissociation of phosphorylated peptide radical cations.

In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [Cu(II)(terpy)(p)M](·2+) and [Co(III)(salen)(p)M](·+) [(p)M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N'-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ((p)M(·+)) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H(3)PO(4)](·+) species through phosphate ester bond cleavage. The CID spectra of the (p)M(·+) species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H(3)PO(4) loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H(3)PO(4) from a prototypical model--N-acetylphosphorylserine methylamide--revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.