Peptide Radical Research Articles

Specific Cα-C Bond Cleavage of β-Carbon-Centered Radical Peptides Produced by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Radical-driven dissociation (RDD) of hydrogen-deficient peptide ions [M - H + H]·+ has been examined using matrix-assisted laser dissociation/ionization in-source decay mass spectrometry (MALDI-ISD MS) with the hydrogen-abstracting matrices 4-nitro-1-naphthol (4,1-NNL) and 5-nitrosalicylic acid (5-NSA). The preferential fragment ions observed in the ISD spectra include N-terminal [a] + ions and C-terminal [x]+, [y + 2]+, and [w]+ ions which imply that β-carbon (Cβ)-centered radical peptide ions [M - Hβ + H]·+ are predominantly produced in MALDI conditions. RDD reactions from the peptide ions [M - Hβ + H]·+ successfully explains the fact that both [a]+ and [x]+ ions arising from cleavage at the Cα-C bond of the backbone of Gly-Xxx residues are missing from the ISD spectra. Furthermore, the formation of [a]+ ions originating from the cleavage of Cα-C bond of deuterated Ala(d3)-Xxx residues indicates that the [a]+ ions are produced from the peptide ions [M - Hβ + H]·+ generated by deuteron-abstraction from Ala(d3) residues. It is suggested that from the standpoint of hydrogen abstraction via direct interactions between the nitro group of matrix and hydrogen of peptides, the generation of the peptide radical ions [M - Hβ + H]·+ is more favorable than that of the α-carbon (Cα)-centered radical ions [M - Hα + H]·+ and the amide nitrogen-centered radical ions [M - HN + H]·+, while ab initio calculations indicate that the formation of [M - Hα + H]·+ is energetically most favorable. Graphical Abstract ᅟ.

Free radical-initiated peptide sequencing (FRIPS)-based cross-linkers for improved peptide and protein structure analysis.

Free radical-initiated peptide sequencing (FRIPS) has recently been introduced as an analytical strategy to create peptide radical ions in a predictable and effective way by collisional activation of specifically modified peptides ions. FRIPS is based on the unimolecular dissociation of open-shell ions and yields fragments that resemble those obtained by electron capture dissociation (ECD) or electron transfer dissociation (ETD). In this review article, we describe the fundamentals of FRIPS and highlight its fruitful combination with chemical cross-linking/mass spectrometry (MS) as a highly promising option to derive complementary structural information of peptides and proteins. FRIPS does not only yield exhaustive sequence information of cross-linked peptides, but also defines the exact cross-linking sites of the connected peptides. The development of more advanced FRIPS cross-linkers that extend the FRIPS-based cross-linking/MS approach to the study of large protein assemblies and protein interaction networks can be eagerly anticipated.

Effect of Hydrogen Bonding and Partial Deprotonation on the Oxidation of Peptides.

In a recent computational study, we found that hydrogen bonding/partial deprotonation facilitates subsequent electron transfer from amides to HO•. We have now analyzed these and related reactions with a glycine derivative as a model peptide, investigating not only reaction energies but also barriers for the individual steps. We find that partial deprotonation not only assists subsequent electron transfer (a sequential proton-loss electron-transfer (SPLET)-type reaction pathway) but also promotes sequential hydrogen-atom transfer (HAT, in a sequential proton-loss hydrogen-atom-transfer (SPLHAT)-type process), both being potential alternatives to direct HAT as routes for peptide oxidation. Each of these alternative pathways is calculated to have energy requirements that make them accessible and competitive. These oxidative processes may produce α-carbon-centered peptide radicals that, through deprotonation, are readily oxidized to the corresponding imines. We have also examined the possibility of competing reactions of amino acid side chains by comparing reactions of the glycine model with those of an analogous valine derivative. We find that, while the side chains of amino acids are more reactive toward direct HAT, a preceding partial deprotonation instead continues to favor the SPLET- and SPLHAT-type reactions, leading to the production of α-carbon-centered peptide radicals. Taken together, these processes have broad implications that impact many aspects of the science and utility of peptides.

Generation and Characterization of Gas-Phase Doubly Charged Biradical Peptide Ions (M2+••).

The gas-phase chemistry of peptide radical ions is attracting considerable interest in the fields of biology and mass spectrometry owing to its capability to provide sequence information on peptides and proteins. In this study, we observed that doubly charged peptide ions (M2+) can be produced from the collision-induced dissociation (CID) of Hg(II)-adducted peptide ions. The chemical nature and, thus, the dissociation pathways of this hydrogen-deficient biradical M2+ species is intriguing. We investigated the generation and dissociation behavior of this M2+ species under electron-capture dissociation (ECD) and CID conditions. The side-chain loss in the CID of the charge-reduced M+• ions formed by single-electron capture suggested that M2+ existed as a biradical ion. This ion underwent the combination of the two radical sites and conversion to hydrogen surplus species through structural rearrangement with increased energies. This study demonstrated a promising method to generate reactive doubly charged biradical precursor ions and, thus, help characterize novel biomolecules.

Tautomerization and Dissociation of Molecular Peptide Radical Cations.

Radical-mediated dissociations of peptide radical cations have intriguing unimolecular gas phase chemistry, with cleavages of almost every bond of the peptide backbone and amino acid side chains in a competitive and apparently "stochastic" manner. Challenges of unraveling mechanistic details are related to complex tautomerizations prior to dissociations. Recent conjunctions of experimental and theoretical investigations have revealed the existence of non-interconvertible isobaric tautomers with a variety of radical-site-specific initial structures, generated from dissociative electron transfer of ternary metal-ligand-peptide complexes. Their reactivity is influenced by the tautomerization barriers, perturbing the nature, location, or number of radical and charge site(s), which also determine the energetics and dynamics of the subsequent radical-mediated dissociatons. The competitive radical- and charge-induced dissociations are extremely dependent on charge density. Charge sequesting can reduce the charge densities on the peptide backbone and hence enhance the flexibility of structural rearrangement. Analysing the structures of precursors, intermediates and products has led to the discovery of many novel radical migration prior to peptide backbone and/or side chain fragmentations. Upon these successes, scientists will be able tobuild peptide cationicanalogues/tautomers having a variety of well-defined radical sites.

Leveraging Electron Transfer Dissociation for Site Selective Radical Generation: Applications for Peptide Epimer Analysis.

Traditional electron-transfer dissociation (ETD) experiments operate through a complex combination of hydrogen abundant and hydrogen deficient fragmentation pathways, yielding c and z ions, side-chain losses, and disulfide bond scission. Herein, a novel dissociation pathway is reported, yielding homolytic cleavage of carbon-iodine bonds via electronic excitation. This observation is very similar to photodissociation experiments where homolytic cleavage of carbon-iodine bonds has been utilized previously, but ETD activation can be performed without addition of a laser to the mass spectrometer. Both loss of iodine and loss of hydrogen iodide are observed, with the abundance of the latter product being greatly enhanced for some peptides after additional collisional activation. These observations suggest a novel ETD fragmentation pathway involving temporary storage of the electron in a charge-reduced arginine side chain. Subsequent collisional activation of the peptide radical produced by loss of HI yields spectra dominated by radical-directed dissociation, which can be usefully employed for identification of peptide isomers, including epimers. Graphical Abstract ᅟ.

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.

Radical Formation Initiates Solvent-Dependent Unfolding and β-sheet Formation in a Model Helical Peptide

We examined the effects of Cα-centered radical formation on the stability of a model helical peptide, N-Ac-KK(AL)10KK-NH2. Three, 100 ns molecular dynamics simulations using the OPLS-AA force field were carried out on each α-helical peptide in six distinct binary TIP4P water/2,2,2-trifluoroethanol (TFE) mixtures. The α-helicity was at a maximum in 20% TFE, which was inversely proportional to the number of H-bonds between water molecules and the peptide backbone. The radial distribution of TFE around the peptide backbone was highest in 20% TFE, which enhanced helix stability. The Cα-centered radical initiated the formation of a turn within 5 ns, which was a smaller kink at high TFE concentrations, and a loop at lower TFE concentrations. The highest helicity of the peptide radical was measured in 100% TFE. The formation of hydrogen bonds between the peptide backbone and water destabilized the helix, whereas the clustering of TFE molecules around the radical center stabilized the helix. Following radical termination, the once helical structure converted to a β-sheet rich state in 100% water only, and this transition did not occur in the nonradical control peptide. This study gives evidence on how the formation of peptide radicals can initiate α-helical to β-sheet transitions under oxidative stress conditions.

Estimation of peptide N-Cα bond cleavage efficiency during MALDI-ISD using a cyclic peptide.

Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) induces N-Cα bond cleavage via hydrogen transfer from the matrix to the peptide backbone, which produces a c'/z• fragment pair. Subsequently, the z• generates z' and [z + matrix] fragments via further radical reactions because of the low stability of the z•. In the present study, we investigated MALDI-ISD of a cyclic peptide. The N-Cα bond cleavage in the cyclic peptide by MALDI-ISD produced the hydrogen-abundant peptide radical [M + 2H](+) • with a radical site on the α-carbon atom, which then reacted with the matrix to give [M + 3H](+) and [M + H + matrix](+) . For 1,5-diaminonaphthalene (1,5-DAN) adducts with z fragments, post-source decay of [M + H + 1,5-DAN](+) generated from the cyclic peptide showed predominant loss of an amino acid with 1,5-DAN. Additionally, MALDI-ISD with Fourier transform-ion cyclotron resonance mass spectrometry allowed for the detection of both [M + 3H](+) and [M + H](+) with two (13) C atoms. These results strongly suggested that [M + 3H](+) and [M + H + 1,5-DAN](+) were formed by N-Cα bond cleavage with further radical reactions. As a consequence, the cleavage efficiency of the N-Cα bond during MALDI-ISD could be estimated by the ratio of the intensity of [M + H](+) and [M + 3H](+) in the Fourier transform-ion cyclotron resonance spectrum. Because the reduction efficiency of a matrix for the cyclic peptide cyclo(Arg-Gly-Asp-D-Phe-Val) was correlated to its tendency to cleave the N-Cα bond in linear peptides, the present method could allow the evaluation of the efficiency of N-Cα bond cleavage for MALDI matrix development. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Theoretical examination of competitive β-radical-induced cleavages of N–Cα and Cα–C bonds of peptides

Selective cleavages of N–Cα and Cα–C bonds of β-radical tautomers of amino acid residues in radical peptides have been examined theoretically by means of the density functional theory at the M06-2X/6-311++G(d,p) level. The majority of the bond cleavages are homolytic via β-scission. Their energy barriers depend largely on the ability of the radical being stabilized in the transition structures and the availability of a mobile proton in the vicinity of the β-radical center. The N–Cα bond is less favorably cleaved than the Cα–C bond (except Ser and Thr) for systems without a mobile proton. It is because, firstly, the homolytic cleavage is less favorable for the more polar N–Cα bond than for the less polar Cα–C bond. Secondly, a less stable σ-radical localized on the amide nitrogen atom of the incipient N-terminal fragment is formed for the former, while a more stable radical delocalized in a π*(CO)-like orbital of the incipient C-terminal fragment is formed for the latter. In the presence of a mobile proton N-terminal to the β-radical center, some degrees of heterolytic cleavage character, as preferred by the polar N–Cα bond, are observed. Consequently, its barrier is reduced. If the mobile proton is located at the C-terminal amide oxygen of the β-radical center, the Cα–C bond cleavage will be significantly suppressed. It is because the radical in the incipient C-terminal fragment becomes more localized as a σ-radical on the carbon atom of its protonated amide group. With basic amino acid residues, the Cα–C bond cleavage can be reactivated. Heterolytic cleavage of the polar N–Cα bond can be largely facilitated if a mobile proton N-terminal to the β-radical center is available and the radical in the incipient C-terminal fragment is sufficiently stabilized, for instance, by the aromatic side chain of Trp and Tyr. Therefore, cleavages of the N–Cα bond induced by the β-radical tautomer of Trp and Tyr are often preferred as compared with cleavages of the Cα–C bond in peptide radical cations containing mobile protons.

Benchmarking Electronic Excitation Energies and Transitions in Peptide Radicals.

Excited electronic states in several radical chromophores representing photochemically active groups in peptide and protein radicals and cation radicals were investigated computationally using equation-of-motion coupled cluster (EOM-CCSD) and time-dependent density functional theory (TD-DFT) methods. The calculations identified the main transitions responsible for photodissociations of gas-phase peptide cation radicals in the near-UV region of the spectrum. Analysis of the EOM-CCSD benchmarks showed that no TD-DFT method was universally accurate across the various radical motifs that included Cα-amide, aminoketyl, formamidyl, guanidyl, carbamyl, benzyl, phenoxy, and tautomeric dihydrophenyl and imidazolyl radicals. Overall, the ωB97XD, M06-2X, and LC-BLYP hybrid functionals showed acceptable performance when benchmarked against EOM-CCSD calculations. However, the performance of these TD-DFT methods depended on the nature of the radical chromophore, emphasizing the need for benchmarking and careful analysis.

OP46 - Superoxide-mediated post-translational modification of tyrosine residues

Superoxide is a byproduct of aerobic respiration. Its in vivo formation is associated with many inflammatory diseases, oxidative stress and aging. Regardless of the rapid spontaneous disproportion of superoxide to give hydrogen peroxide and oxygen, aerobic organisms cannot survive without further catalyzing the rate of this reaction by superoxide dismutase. Therefore, chemical reactions, which contribute to the detrimental effects of superoxide, have to be fast enough to compete with the catalytic dismutation process. The reaction of superoxide with tyrosyl radicals fulfills this criterion and we have shown in a series of publications (see below) that radical addition reactions to tyrosine residues may represent a possible mechanism for superoxide toxicity. Tyr residues are major sites of radical attack on proteins and they also form at active sites of pivotal enzymes (such as cyclo-oxygenase or ribonucleotide reductase) during turnover. This presentation will focus on the mechanisms of the reaction of superoxide with tyrosyl radicals in peptides and proteins in a wide range of model systems. We will present that addition of superoxide to tyrosyl radicals result in the formation of novel Tyr-hydroperoxide species, which can further react with 1) Met residues via intramolecular oxygen transfer to form Met sulfoxide or 2) Cys residues to generate the corresponding disulfide species. These reactions produce a modified bicyclic Tyr monoxide species, which favorably undergo a Michael addition reaction with thiols (such as glutathione) to form novel Tyr-Cys cross-linked adducts.

H-atom loss and migration in hydrogen-rich peptide cation radicals: The role of chemical environment

Hydrogen-atom loss and intramolecular hydrogen-atom migration in peptide radical and cation radical were investigated by quantum chemical calculations for models that include zero to three hydrogen bonds with the ammonium groups. Five different DFT functionals have been checked to study the geometry relaxation and fragmentation processes after electron attachment to peptide cations. The structural changes associated with the geometry relaxation are shown to be dependent on the level of calculation. Furthermore, comparison with CCSD(T) calculations indicates that an adequate description of the electronic state of the reduced protonated peptide is not a sufficient prerequisite to provide accurate energy barrier. Calculating fragmentation reactions of hydrogen-rich peptide cation radicals is pointed out to be a challenging task as none of the DFT functionals are fully effective to describe the whole process. M06-2X is an adequate choice if it provides the correct ground state, otherwise LC-BLYP should be chosen.The electron attachment site and the electron affinity value are highly sensitive to the number of hydrogen bonds with the ammonium group. An ammonium cation with zero, one, and, to a smaller extent, two hydrogen bonds easily captures an incoming electron and subsequently undergoes an H-atom loss process rather than a proton coupled electron transfer. This suggests an explanation for the low amount of c and z fragments observed upon ECD for small protonated peptides, where the ammonium groups cannot be highly intramolecularly “solvated”.

Ultraviolet photodissociation of protonated, fixed charge, and charge-reduced peptides

The fragmentation behavior of three peptides (RGAFSTFGAS, GAFSTFGASR, and GAFSTFGASS) was evaluated by collisional activated dissociation (CAD), higher energy collisional activated dissociation (HCD), and ultraviolet photodissociation (UVPD). In particular, hybrid methods combining electron transfer with CAD, HCD, or UVPD were used to assess the impact of the location of a basic site at the C- or N-terminus, the presence of a fixed charge at the C- or N-terminus, and the presence of a radical site. The release of a mobilized proton occurred for those peptides modified with a quaternary amine at the C- or N-terminus, thus resulting in formation of C- and/or N-terminus fragment ions that would not otherwise be expected if the fixed charge remained static. Activation of charge-reduced peptides (i.e. peptide radical ions) resulted in simplified spectra compared to the corresponding even electron peptides. Interestingly, UVPD of the charge-reduced species generated similar spectra to CAD and HCD of the same precursor ions, suggesting a shift to dominant radical-induced dissociation rather than the more diverse pathways common upon photodissociation.

Specifically designed peptide structures effectively suppressed oxidative reactions in chemical and cellular systems

Peptides were designed based on the identified structural motif of the most potent antioxidant peptides derived from barley protein. Six peptides contained the effective pentapeptide QPYPQ with appended Gln and Pro residues and the other peptides possessed higher hydrophobicity. The peptides have the repetitive sequences of Q, P and Y to investigate the contribution of vicinal residues to their antioxidant capacity. Antioxidant activity of the synthetic peptides was evaluated in chemical and cellular models. Free radical scavenging assays demonstrated the positive role of QP and PY pairs in stabilizing the peptide radicals. Cellular models revealed that repetitive peptide sequences effectively inactivate lipid hydroperoxides and intracellular reactive oxygen species. Peptides with alternating residues were also found effective in inhibition of amyloid fibril formation. These results verified that in addition to the chemical structure of individual side chains, the combined effects of the vicinal residues are also important in antioxidant activity of the whole peptide.

On the formation, stability, and dissociation of peptide radicals after femtosecond electron transfer from alkali metal atoms

In femtosecond encounters between peptide cations and alkali metal atoms, electron transfer occurs from the latter to the former. The peptide radicals are born in the same structure as their precursors and undergo subsequent dissociation on a microsecond to millisecond time scale. The fragmentation pattern is very similar to that observed from other electron-induced dissociation techniques: electron capture dissociation (ECD) where free electrons are captured and electron transfer dissociation (ETD) where the electron donor is an atomic or molecular anion. Dissociation channels involve either (i) loss of a neutral fragment (hydrogen or ammonia) and in some cases consecutive dissociation or (ii) breakage of the peptide backbone, i.e., NCα bond dissociation. We have dubbed the collisional electron transfer technique Electron Capture Induced Dissociation (ECID) to distinguish it from its relatives, ECD and ETD. Here we review ECID results with focus on the competition between dissociation channels and dependence on peptide size and conformation, crown-ether tagging and methylation of ammonium groups, microsolvation, phosphorylation, and the electronic state from which dissociation takes place. While ECID cannot compete with ECD or ETD as an analytical technique, it has provided a wealth of fundamental knowledge on the fragmentation of peptide radicals from work on simple model systems. Its combination with MALDI time-of-flight instruments may, however, show some potential use in the future as ECID does not rely on precursor ions being doubly charged (or higher charged) as neutral fragments can efficiently be converted to anions in secondary collisions.

Transformation of [M + 2H](2+) Peptide Cations to [M - H](+), [M + H + O](+), and M(+•) Cations via Ion/Ion Reactions: Reagent Anions Derived from Persulfate.

The gas-phase oxidation of doubly protonated peptides is demonstrated here using ion/ion reactions with a suite of reagents derived from persulfate. Intact persulfate anion (HS2O8(-)), peroxymonosulfate anion (HSO5(-)), and sulfate radical anion (SO4(-•)) are all either observed directly upon negative nanoelectrospray ionization (nESI) or easily obtained via beam-type collisional activation of persulfate into the mass spectrometer. Ion/ion reactions between each of these reagents and doubly protonated peptides result in the formation of a long-lived complex. Collisional activation of the complex containing a peroxymonosulfate anion results in oxygen transfer from the reagent to the peptide to generate the [M + H + O](+) species. Activation of the complex containing intact persulfate anion either results in oxygen transfer to generate the [M + H + O](+) species or abstraction of two hydrogen atoms and a proton to generate the [M - H](+) species. Activation of the complex containing sulfate radical anion results in abstraction of one hydrogen atom and a proton to form the peptide radical cation, [M](+•). This suite of reagents allows for the facile transformation of the multiply protonated peptides obtained via nESI into a variety of oxidized species capable of providing complementary information about the sequence and structure of the peptide.

Proline as a charge stabilizing amino acid in peptide radical cations

Long distance electron transfer in proteins requires relay stations that can be transitorily oxidized or reduced. Although individual prolines cannot assume this function, because of their high ionization energy, it has been shown that polyprolines have the ability to transfer charges. In order to determine the role of the proline in the hole distribution and transport within a PheProPhe tripeptide, the radical cation of a model compound where the phenylalanines carry two or three methoxy groups, respectively, was generated by flash photolysis. Surprisingly, after equilibration, about two thirds of the holes were found to reside on the phen(OMe)2 instead of the more easily oxidizable phen(OMe)3 moiety. DFT calculations showed that, in most of the accessible conformations, the phen(OMe)2• +‐moiety profits more from stabilization by N‐ and/or O‐lone pairs of neighboring amide groups than the phen(OMe)3• + moiety can, which explains the apparently counterthermodynamic hole distribution. Similar calculations showed that, in several conformers of the natural PheProPhe radical cation, the unpaired electron is delocalized over two amide groups, by residing in a σ MO which links the N‐lone pair of the central proline unit with the O‐lone pair of a proximate amino acid, through hyperconjugation via the intervening C―Cα σ‐bond. The same pattern is found in a model compound, N‐acetylproline dimethylamide. It seems that prolines favor conformers which foster hyperconjugation of two amide groups, which lowers the ionization energy of peptides. One should thus consider such interacting amide groups as potential relay stations in the course of electron transfer in polyprolines. Copyright © 2015 John Wiley & Sons, Ltd.

Combining UV photodissociation with electron transfer for peptide structure analysis.

The combination of near-UV photodissociation with electron transfer and collisional activation provides a new tool for structure investigation of isolated peptide ions and reactive intermediates. Two new types of pulse experiments are reported. In the first one called UV/Vis photodissociation-electron transfer dissociation (UVPD-ETD), diazirine-labeled peptide ions are shown to undergo photodissociation in the gas phase to form new covalent bonds, guided by the ion conformation, and the products are analyzed by electron transfer dissociation. In the second experiment, called ETD-UVPD wherein synthetic labels are not necessary, electron transfer forms new cation-peptide radical chromophores that absorb at 355 nm and undergo specific backbone photodissociation reactions. The new method is applied to distinguish isomeric ions produced by ETD of arginine containing peptides.