Peptide Radicals and Cation Radicals in the Gas Phase - Chemical

Review pubs.acs.org/CR

Peptide Radicals and Cation Radicals in the Gas Phase František Tureček* Department of Chemistry, Bagley Hall, University of Washington, Seattle, Washington 98195-1700, United States

Ryan R. Julian Department of Chemistry, University of California, Riverside, California 92521, United States 7.3. Peptide Dissociation Pathways and Mechanisms 7.3.1. Backbone Dissociation 7.3.2. Side-Chain Losses 7.3.3. Summary of Hydrogen-Deficient Peptide Cation Radicals 8. Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 1.1. Scope 1.2. Early Research on Peptide Cation Radicals 2. Hydrogen-Rich and -Deficient Peptide Radicals and Cation Radicals 3. Generation of Hydrogen-Rich Peptide Radicals and Cation Radicals 4. Energetics of Peptide Radical and Cation Radical Formation 5. Mechanisms of Peptide Radical and Cation Radical Formation 5.1. Dipolar Effects on Electron Attachment 6. Dissociations of Hydrogen-Rich Peptide Radicals and Cation Radicals 6.1. Loss of H Atoms 6.2. Loss of Ammonia 6.3. Disulfide Bond Cleavage 6.4. Peptide Backbone N−Cα Bond Cleavage 6.4.1. Mechanisms of N−Cα Bond Cleavage 6.4.2. Effects of Peptide Ion Conformation 6.4.3. Backbone Cleavage Frequency 6.5. Postcleavage Ion−Molecule Reactions of c and z fragments 6.6. Loss of Side-Chain Groups 6.7. Rearrangements in Peptide Radicals and Cation Radicals 6.8. Modified Peptide Cation Radicals 6.8.1. Fixed-Charge Groups 6.8.2. Radical and Electron Traps 6.8.3. Crown Ether and Other Noncovalent Peptide Complexes 6.8.4. Peptides with Modified Backbones 6.9. Peptide−Metal Complexes 7. Hydrogen-Deficient Peptide Cation Radicals 7.1. Radical Generation 7.1.1. Collisional Activation 7.1.2. Photoactivation 7.2. Radical Migration © 2013 American Chemical Society

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1. INTRODUCTION For about three decades, the chemistry of cation radicals derived from organic molecules had received a major impetus from mass spectrometry. This was because cation radicals were readily obtained by electron impact ionization of closed-shell organic molecules, and the various mass spectrometric techniques available to researchers allowed them to study transient species with lifetimes on the nano- to microsecond time scale. Much of unimolecular cation radical chemistry, including thermochemistry,1 kinetics,2 and stereochemistry,3−5 has been explored by mass spectrometry to aid the rational interpretation of ion fragmentations.6 The chemistry of gasphase cation radicals showed some analogies with their chemistry in solution, for example, in cycloaddition or cycloreversion reactions,7−9 but also produced quite unusual species that do not have stable condensed phase equivalents, such as distonic ions.10−12 The salient aspect of cation radical reactions is that they are mostly radical driven; that is, they involve homolytic bond cleavages and hydrogen atom migrations.6 Mass spectrometry of small free radicals has been reviewed.13 With the advent of the so-called soft-ionization methods, starting with 252Cf plasma desorption14 and fast atom bombardment,15 and culminating with electrospray16 and matrix-assisted laser desorption17,18 ionization (MALDI), the focus has shifted to the chemistry of gas-phase even-electron ions with closed electron shells, which are chiefly produced by these ionization methods. Simultaneously, the increased use of ion trapping techniques in mass spectrometry has led to a shift

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Received: January 24, 2013 Published: May 7, 2013 6691

dx.doi.org/10.1021/cr400043s | Chem. Rev. 2013, 113, 6691−6733

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interest. Section 3 covers the experimental methods for the generation of hydrogen-rich peptide cation radicals, and section 4 summarizes the energetics of electron attachment and transfer. Section 5 then deals with the quantum-chemical aspects of electron attachment and the electronic states involved in this process. Section 6 addresses in detail the various types of dissociations induced by electron attachment to peptide cations. Finally, section 7 covers the chemistry of hydrogen-deficient peptide cation radicals and provides some links to the chemistry of hydrogen-rich ions.

of the observation time for ion formation and reactions from low microseconds to high milliseconds, changing the kinetic window accessible by routine mass spectrometers. Last but not least, ion activation methods in ion traps operate in the “slow heating” regime,19 which tends to sample ion dissociations following lowest activation energy pathways. Dissociations of even-electron ions under slow heating conditions are dominated by heterolytic bond cleavages accompanied by proton or larger group migrations that often result in quite complicated reaction pathways. In the particular case of peptide even-electron ions, the main dissociations are eliminations of small molecules (water, ammonia) and proton-driven cleavages of amide bonds. The latter are essential for peptide sequencing by mass spectrometry.20 It is useful to introduce here the standard notation of peptide backbone fragments according to Roepstorff and Fohlman21 and Biemann,22 which is shown in Scheme 1. The N-terminal

1.2. Early Research on Peptide Cation Radicals

The early attempts to generate peptide cation radicals required special laser techniques for sample evaporation and ionization, as well as the presence of a suitable chromophore in the peptide molecule. Cation radicals of the (peptide)+• type were first generated by Schlag, Grotemeyer, and their co-workers using resonance-enhanced multiphoton ionization (REMPI) of laserdesorbed peptides in the gas phase.32−36 In these experiments, a solid peptide was desorbed by a laser pulse and entrained in a pulse of cold argon atoms from a supersonic expansion jet. The entrained molecules were cooled by collisions with Ar atoms and carried by the gas plume to intercept the ionizing laser also operated in a pulse mode. Because of the need of a chromophore for REMPI, this method was limited to peptides containing aromatic residues (phenylalanine, tyrosine, or tryptophan). A different approach to peptide cation radicals relied on transition metal complexes produced by electrospray that showed radical-driven dissociations such as homolytic bond dissociations upon collisional activation in the slow heating regime.37−40 With a proper choice of the metal ion and organic ligands, peptide ternary complexes have been shown to undergo intramolecular electron transfer upon collisional activation, producing metal-free peptide ions.41,42 Collisioninduced dissociation (CID) of ternary peptide−transition metal complexes with an auxiliary organic ligand has been used for the production of (peptide)+• cation radicals, as pioneered by Siu and co-workers.43−48 The complexes self-assemble in solution and are brought to the gas phase by electrospray ionization.37−41 The gas-phase complexes are selected by mass and stored in an ion trap. Collisional activation results in a dissociation involving intramolecular electron transfer to form (peptide)+• ions. Siu and co-workers originally used copper complexes, which allowed for the efficient formation of peptide cation radicals only from peptides containing readily ionizable aromatic residues.43,44 Further refinement of auxiliary ligands in copper complexes,45−49 as well as the use of other transition metal complexes,50,51 broadened the scope of peptide cation radicals that can be generated this way. This topic has been recently covered in extensive reviews by Hopkinson and Siu.52,53 It is worth noting that direct ionization of laserdesorbed peptides and CID of peptide−metal complexes in general do not produce the same peptide cation radicals, because CID can be accompanied by skeletal or hydrogen rearrangements. Peptide cation radicals of the (peptide + nH)(n−1)+• and (peptide)+• types differ in their electronic structure and reactivity and therefore will be treated separately. More recently, thermal or photochemical dissociation of suitable auxiliary groups has been used to generate radical centers in gas-phase peptide ions that can be then studied by mass spectrometry. These methods and the cation radicals produced by them will be covered in Section 7. These various

Scheme 1

fragments are denoted by am, bm, and cm where m is the number of amino acid residue α carbons in the fragment ion. The complementary C-terminal fragment ions are denoted by xk, yk, and zk, where n = k + m is the number of amino acid residues in the parent peptide ion. Note that Scheme 1 does not attempt to assign concrete structures to the fragment ions. Dissociations of even-electron peptide ions typically, albeit not uniformly, produce b and y type ions, which are favored by enthalpy or entropy.23,24 Sometimes, this represents a limitation of peptide even-electron ion dissociations, especially when the peptide contains residues such as proline or aspartic acid that highly favor backbone cleavage in their vicinity and thus competitively suppress other peptide bond dissociations with deleterious results for sequence determination.25−28 This feature is of a fundamental nature and cannot be circumvented by means other than entirely changing the peptide chemistry. Furthermore, because of the presence of multiple nucleophilic groups in peptide cations, the backbone dissociations are often accompanied by cyclizations and rearrangements that may obscure the sequence readout.29−31 1.1. Scope

The topic of this review is the gas-phase chemistry of peptide radicals and cation radicals. These transient species have become available owing to the development of new techniques of ion formation and activation, and because of their utility in peptide and protein sequencing, they represent a rapidly growing field of interest in chemistry and biology. Our understanding of their rather novel chemistry is continuously developing, and the goal of this review is to both capture the current state of the art in a systematic fashion and provide hindsight relating to how the current ideas have come about. The review is organized as follows. In this section (section 1), we provide a brief overview of the earlier research on generating peptide cation radicals. Section 2 presents a general definition of hydrogen-rich and hydrogen-deficient peptide cation radicals, which are the two basic types of species of 6692


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+ nH)n+ where n is the charge state. Hydrogen-rich peptides are typically produced by attachment of an electron to a multiply protonated peptide ion, fitting the formula (M + nH)(n−1)+•. Conversely, hydrogen-deficient peptide cation radicals are those produced by abstraction of a hydrogen atom or a radical group from a protonated peptide ion or by loss of an electron from neutral peptide. Here the formula is (M + (n − 1)H)n+•, which becomes M+• for n = 1. The reasons for distinguishing hydrogen-rich and -deficient peptide cation radicals are their different methods of generation, electronic states, and reactivity. Sections 3−6 of this review deal with the generation, electronic properties, and dissociations of hydrogen-rich peptide radicals and cation radicals. Section 7 then deals with the aspects of generation and chemistry of hydrogen-deficient peptide cation radicals.

methods produce peptide cation radicals of different types that are as a rule sorted into two groups called hydrogen-rich and hydrogen-deficient ions.

2. HYDROGEN-RICH AND -DEFICIENT PEPTIDE RADICALS AND CATION RADICALS The term hydrogen-rich is straightforwardly applied to a neutral peptide molecule (M) and simply implies addition of a hydrogen atom, (M + H)•. Conversely, abstracting a hydrogen atom from the neutral peptide molecule forms a hydrogendeficient peptide radical (M − H)• (Scheme 2). The situation Scheme 2

3. GENERATION OF HYDROGEN-RICH PEPTIDE RADICALS AND CATION RADICALS A major breakthrough in this area came with the discovery that recombination of low-energy electrons with multiply charged peptide and protein ions produced abundant fragment ions that carried information on the amino acid sequence. Figure 1 illustrates the wealth of information carried by the numerous backbone fragments present in the spectrum of ubiquitin (M + 11H) 11+ ion after ion−electron recombination. 54 This technique called electron capture dissociation (ECD)55 opened an avenue to the convenient formation of peptide and protein cation radicals in a Penning trap of a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The various aspects of ECD instrumentation have been reviewed,56−58 and

is somewhat less straightforward with peptide cation radicals because the charges are typically provided by protonation, which increases the number of hydrogen atoms in the ion relative to the neutral peptide molecule, that is, (M) versus (M

Figure 1. Electron capture dissociation mass spectrum of [ubiquitin + 11H]11+ ion. Adapted with permission from ref 54. Copyright 2000 American Chemical Society. 6693


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results in extensive dissociation, as reported for a few dipeptides,86−88 and does not appear to be particularly useful for studying peptide radicals. In contrast, collisional reionization to anions produces stable anions only if the radical intermediate has a bound state of a microsecond lifetime for the incoming electron. This feature has been used for structure assignment of peptide radicals, as discussed in section 6.7 below.

most modern commercial FT-ICR mass spectrometers are equipped with an external electron source for ECD measurements. For other techniques employing ion−electron reactions in ion traps, see refs 59−63. An alternative method for the production of cation radicals of the same type is by collisional electron transfer from a suitable donor. This can be a neutral atom or molecule (N), as in electron capture-induced dissociation (ECID),64,65 or a molecular anion radical (A−•), as in electron transfer dissociation (ETD).66−68 ECID is typically performed with mass-selected ion beams on sector instruments at ion kinetic energies ranging from 3 keV for the instrument in Osaka65 to 100 keV for the instrument in Aarhus.64 Both of these are home-built or modified instruments. ETD is far more common and commercially available in many ion trap mass spectrometers equipped with external chemical ionization sources for the production of anion reactants.67−69 Fluoranthene has been commonly used, because it readily forms an anion radical that undergoes efficient electron transfer to peptide ions. Other reagents have been used, for example, anthracene,66 SO2,70,71 azobenzene,72 azulene,73 nitrosobenzene, and 1,4-dicyanobenzene. Research instruments have been developed that use other sources of anions, such as a glow discharge source,70,74 or one using dual spray and alternating high voltage potentials has been used for the production of cations and anions.74,75 Recently, ETD in an ion beam has been introduced by coflowing radical anions and peptide ions through stacked76 or multipole electrostatic lenses.77 ECD, ETD, and ECID are sometimes referred to under the common acronym of ExD. The common feature of all the ExD methods is that they produce peptide radicals or cation radicals that formally arise by hydrogen atom addition to a neutral (n = 1) or protonated (n ≥ 2) peptide (eqs 1−3). (peptide + nH)n + + e− → (peptide + nH)(n − 1) +•

4. ENERGETICS OF PEPTIDE RADICAL AND CATION RADICAL FORMATION Owing to the nature of mass spectrometric measurements, reactions of gas-phase radicals and cation radicals are most often observed as unimolecular dissociations of isolated species. Under these conditions and given the short experimental time scales, the reactant internal energy is conserved and is typically determined by the ion formation process. At longer lifetimes pertinent to ions trapped in an ICR cell, the ion internal energy can be depleted by radiative cooling.89,90 The energetics of peptide radicals differ depending on the mode of radical formation. Capture of a free electron can overall be described by a thermochemical cycle (Scheme 3), where RE is the ion− Scheme 3

(1)

electron recombination energy that defines the electron capture exothermicity, PA and Haff are, respectively, the proton affinity and hydrogen atom affinity of the lower charge state, and IEH is the ionization energy of the hydrogen atom (13.59 eV). Scheme 3 has been used for rough estimates of ion−electron recombination energies.56,57 Its use is limited because proton affinities of peptide ions are mostly unknown and difficult to measure.91 Nevertheless, Scheme 3 allows one to estimate the trends in peptide ion recombination energies as a function of proton affinity. For example, intramolecular hydrogen bonding of the charged group is supposed to stabilize the ion and increase the proton affinity of the lower charge state, resulting in a decrease of recombination energy. A more specific approach to the energetics of ion−electron recombination considers the stepwise interaction of the electron with the electrostatic field of the peptide cation, which results in a cascade of developing electronic states of the charge-reduced species (Figure 2). Electron capture forming a hypothetical Nth electronic state is associated with an adiabatic recombination energy, REa = E(ion) − E(N), which can be expressed as the sum of the vertical recombination energy to reach the N state (REv(N)) and a Franck−Condon energy (EFC). The recombination energy is dissipated by intramolecular vibrational energy redistribution (IVR) among the vibrational degrees of freedom of the N state. This electronic state can cross to a lower state (M) if the two states have substantial vibronic coupling.92 However, the state crossing

(peptide + nH)n + + A−• → (peptide + nH)(n − 1) +• + A (2)

(peptide + nH)n + + N → (peptide + nH)•(n − 1) + + N+• (3)

Although most work to date has been done with protonated peptides, it should be noted that one or more charging particles could be alkali or transition metal cations, which also readily attach to peptides by electrospray ionization (see Section 6.9). Electron attachment to multiply charged ions according to eqs 1 and 2 is typically carried out in an ion trap, which allows sequential capture of two or more electrons resulting in lower charge states of the transient radical intermediates. A specific case concerns collisional electron transfer at kiloelectronvolt kinetic energies (eq 3), which is performed on sector mass spectrometers working with fast ion beams. Electron transfer takes place in glancing collisions with alkali metal atoms at extremely short duration (10−15−10−14 s) and under experimental conditions favoring single collisions and transfer of one electron. Another specific feature of eq 3 processes concerns discharge of singly protonated peptides (n = 1) to form neutral (peptide + H)• radicals. These and their neutral dissociation products cannot be directly detected by mass spectrometry but must be reionized by collisional electron detachment to form cations78−83 or by collisional electron transfer to form anions.84,85 Reionization to cations frequently 6694


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Table 1. Recombination Energies of Peptide Cations recombination energya ion +

REa

[Arg + H] [Arg-NH2 + H]+ [His-NHCH3 + H]+ [Nα-Ac-His-NHCH3 + H]+ [Gly-His + H]+ [His-Gly + H]+ [Gly-Arg + H]+ [Gly-Lys + H]+ [Pro-Gly + H]+ [Gly-Pro + H]+ [Gly-Gly-NH2 + H]+ [His-Ala-Ile + H]+ [His-Ala-Leu + H]+ [Ala-His-Leu + H]+ [Ala-Leu-His + H]+ [His-Asp-Ala-Ala-Leu + H]+ [His-Ala-Asp-Ala-Leu + H]+ [Ala-His-Asp-Ala-Leu + H]+ [Ala-His-Ala-Asp-Leu + H]+

Figure 2. Energy diagram for electron attachment to a gas-phase ion.

competes with dissociations occurring from the particular electronic state of the charge-reduced species. After going through a cascade of state crossings combined with IVR, the charge-reduced species can reach the ground state (X) in which the ion recombination energy has been converted to vibrational excitation. With one exception, recombination energies for singly and doubly protonated peptides have not been measured, and the majority the available data come from ab initio and density functional theory calculations that provide local energy minima of the ground electronic states. The exception was the measurement of the recombination energy in a multiply hydrated Lys-Tyr-Lys dication by Williams and co-workers, which relied on counting the desorbed water molecules to estimate the internal energy deposition upon electron capture.93 Representative data from the computational studies are collated in Tables 1 and 2. Singly protonated amino acids and di- and tripeptides show a remarkably narrow range of adiabatic recombination energies of 3.2−3.7 eV. There is no strong dependence on the nature of the protonation site, which is the guanidine group in arginine residues, imidazole ring in histidine residues, ε-amino group in lysine residues, and the N-terminal amino or amide group in peptides lacking basic residues. The REa decreases with the size of the peptide ion, for example, from 3.4 eV in the histidinecontaining tripeptides to 2.8 eV in the pentapeptides (Table 1; refs 94−100). This is likely due to the stabilization of the charged histidine group by the neutral dipoles of the carboxyl and amide group. In contrast, hydrogen bonding of the histidine imidazole protons, which is different in the (His-AlaLeu + H)+ and two stable conformers of the (His-Ala-Asp-AlaLeu + H)+ ion (Figure 3), has only a minor effect. The recombination energies (adiabatic REa and vertical REvert) of doubly protonated pentapeptides were calculated to be in a 4.5−5.7 eV range and depended more strongly on the peptide ion sequence, amino acid composition, and ion conformation (Table 2).101−107 For example, the low-energy globular conformer of (pSer-Ala-Ala-Ala-Erg + 2H)2+ was calculated to have a higher recombination energy than the extended conformer of (Ala-pSer-Ala-Ala-Arg + 2H)2+.105 Peptide ions with no basic groups, for example, (Ala-Ala-βAbAla-Ala + 2H)2+ (Table 2),106 showed notably higher

c

3.37−3.40 3.49−3.53c 3.46 3.63−3.74c 3.49−3.64c 3.48 3.42−3.75c (4.24)d 3.36 (4.31)d 3.71 3.27 3.30 3.45 3.31 2.83 2.96 2.93 3.07

REvb

ref

2.19 2.67 2.62 2.67−2.79c 2.61 2.79 2.64−2.81c 3.15 3.07 3.17 2.77 2.29 2.39 2.46 2.43 2.20 2.16−2.30c

94 95 96 96 96 96 97, 98 98 99 99 98 100 100 100 100 100 100 100 100

2.21

a

In units of electronvolts. From combined B3LYP and PMP2 calculations with the 6-311++G(2d,p) and 6-311++G(3df,2p) basis sets and including zero point energy corrections. bThe vertical recombination energies do not include zero-point energy corrections. c For different ion and radical conformers. dRadicals isomerized upon electron attachment.

recombination energies than those containing protonated arginine, lysine, or histidine residues. This effect is likely to depend on the precursor peptide ion conformation, which determines the spatial arrangement of the charged groups and thus the Coulomb energy that is relieved upon electron attachment. However, no systematic study of peptide ion conformations is available so far to generalize this effect. Differences in the REa and REv are due to two effects. One is Franck−Condon effects in electron attachment to the planar πsystems in histidine108 and arginine95 that relax by pyramidization as the radicals reach the ground state geometry. The other effect is due to exothermic isomerizations by proton migration, which lowers the potential energy of the charge-reduced ions and thus increases the adiabatic recombination energy. Extending the peptide ion sequence from pentapeptides to hepta- and nonapeptides results in a slight decrease of the recombination energy, which converges to ca. 4 eV for the REvert.107 This effect can be ascribed to a combination of reduced Coulomb repulsion and increased polarizability in the larger ion. This appears to be consistent with the only experimental measurement of recombination energy in a highly hydrated Lys-Tyr-Lys dication that gave RE = 4.3 eV.93 Increasing the charge from +2 to +3 in a heptapeptide ion increases the REvert by about 1.3 eV (Table 2). Electron transfer to peptide ions from neutral or anion donors has some additional specific features that reflect the donor−acceptor interaction and the nature of the electron transfer process. Electron transfer from a neutral donor, typically an alkali metal atom, occurs between a fast ion beam and thermal neutral atoms, which limits the ion−donor interaction to several femtoseconds. This is expressed by eq 4, where m is the precursor ion mass, σ is the cross section for electron transfer, and U is the ion kinetic energy. 6695


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⎡ (ze)2 α(z + 1)2 e 2 ⎤ ⎥ ΔE = RE vert(ion) − IEmetal − ⎢ + 32π 2ε0 2R c 4 ⎦ ⎣ 4πε0R c

Table 2. Recombination Energies of Peptide Di- and Trications recombination energyb ion

a

(Gly-Lys + 2H)2+ (Lys-Lys + 2H)2+ (Gly-Gly-Lys + 2H)2+ (Gly-Arg + 2H)2+ (Ala-Arg + 2H)2+ (Ala-Ala-His-Ala-Leu + 2H)2+ (Ala-His-Asp-Ala-Leu + 2H)2+ (Ala-His-Ala-Asp-Leu + 2H)2+ (Ala-Ala-Val-Ala-Arg + 2H)2+ (Ala-Ala-Asp-Ala-Arg + 2H)2+ (Ala-Ala-His-Ala-Arg + 2H)2+ (Lys-Tyr-Lys + 2H + nH2O)2+ (pSer-Ala-Ala-Ala-Arg + 2H)2+ (Ala-pSer-Ala-Ala-Arg + 2H)2+ (Ala-Ala-pSer-Ala-Arg + 2H)2+ (Ala-Ala- Ala-pSer-Arg + 2H)2+ (Ala-Ala-βAb-Ala-Ala + 2H)2+ (Ala-Ala-βAb-Ala-Ala + 2H)2+ (Leu-Lys-Gly-Pro-Ala-AspArg + 2H)2+ (Leu-Lys-Gly-Pro-Ala-AspArg + 3H)3+ melectin fragment2+g

(5)

REvc

ref

5.77 (6.12−6.36)e 5.46 (5.79−6.59)e 6.2c

5.50−5.65d 4.80−4.95d 4.94 4.96−5.31d 4.9−5.0

5.13−5.46d

4.45−4.72d

101 101 98 97 97, 103 102

5.40−5.53d

4.45−4.52d

102

5.29−5.47d

4.52

102

5.54−5.61d 4.77−4.84

4.80

104 104

4.97 4.3 (n = 12−25)f

4.0

104 93

5.77−5.99d,e

4.33−4.81d

105

4.53−4.88d

4.24−4.30d

105

5.44−5.50d,e

4.36−4.40d

105

d,e

d

105

REa

5.46−5.75

4.51−4.60

6.53

5.34

106

5.68

4.83

106

5.23e

4.02−4.29d

5.94−6.28

5.36−5.79d 4.0

It was argued that ion-induced dipole interaction is negligible at typical Rc values, and thus eq 5 can be rewritten in a simplified form (eq 6), where σ is in Å2 (10−16 cm2).102 ΔE(eV) ≅ RE vert(ion) − IEmetal −

107

The protonated residues are shown in bold. bIn units of electronvolts. From combined B3LYP and PMP2 calculations with the 6-311+ +G(2d,p) and 6-311++G(3df,2p) basis sets and including zero point energy corrections. cThe vertical recombination energies do not include zero-point energy corrections. dFor different ion and radical conformers. eRadicals isomerized upon electron attachment. fExperimental value. g(Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-NH2 + 2H)2+.

t=

(6)

A positive ΔE in eqs 5 and 6 means that the electron transfer is exoergic. The repulsion term vanishes in electron transfer producing neutral peptide radicals and is replaced by ion-dipole and ion-induced dipole terms for the attractive interaction between the neutral peptide radical and the metal cation that separate in the exit channel. These terms slightly reduce the ΔE. It is noteworthy that alkali metal cations have excitation energies >10 eV for the np → (n + 1)s electronic transitions and thus a very low effective “heat capacity” for excess energy partitioning. Hence, the ΔE is deposited as vibronic excitation in the charge-reduced peptide. The fundamental aspects of electron transfer from anions were studied by McLuckey and co-workers who put forth a semiempirical model based on Landau−Zener theory.110 The important issue in cation−anion reactions is the competition between the undesirable proton transfer and desirable electron transfer. Both processes are highly exothermic for organic cations in general. With multiply charged peptide cations, proton transfer is energetically favored because the gas-phase acidities of donor conjugate acids exceed the gas-phase basicity of the lower charge state of the peptide ion. The probability for electron transfer, PET, depends on the Landau−Zener probability, PLZ, for nonadiabatic avoided crossing from the potential energy surface of the reactants, which are the multiply charged peptide cation and the electron donor anion radical, to the surface of the products, which are the charge-reduced peptide cation radical and the neutral molecule of the electron donor, according to eq 7, which has a maximum at PLZ = 0.5. This ensues from crossing and recrossing the avoided crossing region on the reactant and product curves with PLZ(1 − PLZ) probability for electron transfer in each event.

a

2mσ πU

25.52 σ

PET = 2PLZ(1 − PLZ)

(7)

PLZ further exponentially depends on the vibronic coupling element of the surfaces, relative reactant velocity, and reactant separation at the avoided crossing point (rET). The estimated rET scales inversely with the reaction exothermicity, −ΔHET = RE(ion) − EA(donor) > 0, but is substantially greater than the reactant distance in the transition state for proton transfer. Another feature of electron transfer in an ion−ion reaction is that the excess energy can be partitioned between the chargereduced peptide ion and the neutral molecule of the electron donor. The partitioning can take the form of vibrational and electronic excitation such that the excitation energy (ΔE) in the charge-reduced peptide ion can be written as

(4)

For example, in electron transfer to a peptide ion of m = 1000 Da, U = 105 eV, and σ = 10−13 cm2, the interaction time is limited to 25 fs. The energy balance (ΔE) in electron transfer can be expressed by eq 5, which involves the vertical recombination energy of the peptide ion (REvert), the ionization potential of the atomic donor (IEmetal), the repulsive Coulomb potential between the charge-reduced peptide ion and the alkali metal ion separating after electron transfer, and the attractive ion-induced dipole between the precursor ion and the alkali metal atom entering the collision.105,109 Rc is the critical distance between the electron donor and acceptor at which electron transfer occurs, which is related to the cross section σ, Rc = (σ/π)1/2, and α is the metal atom polarizability.

ΔE = RE(ion) − EA(donor) − Eexc

(8)

where Eexc is the excitation of the neutral reaction product. The electron donors used in ETD are typically aromatic anion radicals that have favorable Franck−Condon factors for transition to the corresponding neutral molecules. Thus, the transition is associated with low vibrational excitation in the 6696


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Figure 3. Structures of low-energy ion conformers of (His-Ala-Leu + H)+ and (His-Ala-Asp-Ala-Leu + H)+ from M06-2X/6-31+G(d,p) calculations. Data from ref 99.

⎛ 2Z ⎞ ⎟ σET = PETπrET 2⎜1 + rETμvrel 2 ⎠ ⎝

neutral aromatic molecule, and the Eexc term in eq 8 mainly represents electronic excitation in the aromatic π* system. Figure 4 shows generic surface crossings for electron transfer

(9)

The overall reaction exothermicity (ΔE = 4.5 eV for electron transfer to a doubly charged peptide)102 is used to excite the charge-reduced peptide ion. However, ET hopping to curves leading to excited electronic states of fluoranthene (S1, S2; Eexc = 3.45 and 4.32 eV, respectively)111,112 leaves much less energy to be used to excite the charge-reduced peptide ion. In addition, these avoided crossings occur at larger rET and, depending on the respective PET, can lead to substantial cross sections for these channels. Thus, ET forming fluoranthene or other aromatic electron donor molecules in the S0, S1, and S2 states presumably results in multimodal energy distribution in the charge-reduced peptide ion. The actual internal energy distribution in charge-reduced peptide ions produced by ET is difficult to measure and can be further modified by collisions with the background gas in the ion trap.

5. MECHANISMS OF PEPTIDE RADICAL AND CATION RADICAL FORMATION One-electron reduction of peptide ions, both by electron capture and transfer, raises the question of the nature of the electronic states being generated, their lifetime, and reactivity. An early paper on ECD suggested that ion−electron recombination initially forms a high Rydberg electronic state that decays by crossing to a repulsive valence σ* state and results in prompt backbone bond dissociation.113 This was modeled on dissociative recombination of small singly charged ions where dissociative states had been identified by rigorous analysis and molecular spectroscopy.114 Another motivation for considerations of prompt dissociation was the finding that even protein ions showed substantial dissociation upon electron capture that was deemed improbable given the large number of internal degrees of freedom into which the initial excitation should be partitioned. DFT calculations on a small model system, the CH3C(OH)NHCH3 radical, were taken as evidence that protein radical intermediates should undergo backbone cleavage in preference

Figure 4. Approximate potential energy curves for electron transfer from an anion radical to a peptide dication. The blue curve depicts the reactant potential energy surface, the red and black curves represent the product channels for various electronic states of the chargereduced peptide (X, A) and neutralized electron donor (S0, S1).

from the fluoranthene anion radical (EA = 0.6 eV).111 The blue curve depicts the attractive potential between the peptide dication and [fluoranthene]−• that reaches avoided crossings with the curves for the separation of the ET products, which are the charge-reduced peptide ion and neutral fluoranthene molecule. Avoided crossing with the ground electronic state of the ET products (red curve in Figure 4) at a distance of rET gives a cross section (σET), which is expressed by eq 9, where PET is the electron transfer probability from eq 7, Z is the peptide ion charge, μ is the reduced mass, and vrel is the reactant relative velocity. This leaves the fluoroanthene molecule in the ground singlet state (S0) of Eexc = 0. 6697


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Coulomb law (eq 10), where ΔE is the electron binding energy and R is the distance between the charges in angstroms.

of loss of hydrogen atom. Loss of hydrogen atoms was claimed to be absent in protein ECD.113 A more thorough investigation of the CH3C(OH)NHCH3 radical by experiment115,116 and analysis of the potential energy surface by high-level (G2MP2) calculations115 proved the previous claims incorrect.115,117,118 The radical and several of its deuterium-labeled analogues were generated by collisional electron transfer from well-defined precursor ions and found to competitively cleave the N−Cα bond and eliminate the H atom, which mainly originated from the OH group. The experimental branching ratio for these two channels (1:1.7) was closely matched by theoretical rate constants from RRKM calculations on the G2(MP2) potential energy surface. The claim of no H-atom loss in protein ECD113 was later abandoned,117 and instead an abundant loss of H was reported for ECD of ubiquitin ions and suggested to be indicative of the gas-phase protein ion folding.119 The early views of peptide and protein cation radicals presumed that ion−electron recombination involved electron attachment to the charged groups. For ammonium groups, electron−ion recombination in simpler organic ammonium ions was known to result in cleavages of the N−H and C−NH3 bonds,120−133 which bore resemblance to loss of H and ammonia upon ECD. However, the role of other chargecarrying groups such as arginine guanidininum and histidine imidazolium was not considered in this early model. A previous study of electron transfer to benzylammonium cations concluded from the observed dissociations that the electron entered the π* orbitals on the aromatic ring carrying electronegative fluorine or nitro substituents, forming zwitterionic intermediates in which the ammonium group retained the positive charge and the introduced unpaired electron was delocalized over the negatively charged aromatic ring.128 A cogent model for electron attachment to peptide ions was independently proposed by Simons134 and Turecek and their co-workers135 that considered electron attachment to the π* orbitals of the peptide amide groups. Note that neutral amide groups have no bound electronic states for electron attachment and hence they have negative electron affinities. However, the Coulomb effect of the positive charges in the peptide ion stabilizes the π* states of amide anion radical intermediates so that they can represent bound electronic states. An early paper by Simons and co-workers addressed the question of the electronic states accessed by electron attachment to neutral Ala-Ala dipeptide furnished with two remote point charges placed at 10 Å from the molecule.134 Since, in the absence of symmetry restrictions, Hartree−Fock calculations of excited states undergo variational collapse to the ground electronic state, Simons et al. used the method of Peyerimhoff et al.114,136,137 that was designed for loosely bound anions. By artificially increasing the nuclear charge, the unpaired electron is kept in orbitals close to selected atoms and then the calculations are extrapolated to normal nuclear charge. Attachment of an electron to any of the σ* bonds resulted in repulsive potential energy curves, but showed no specific reactivity. In contrast, electron attachment to the amide π* orbital led to bound states, and cleavage of the adjacent N− C and C−C bonds showed different potential energy barriers. An estimate was made that a neutral amide can have a bound state for an electron if furnished with a point positive charge at a distance not exceeding 6 Å.134 This followed from an energy estimate for the virtual state of the neutral amide (∼2.5 eV from an electron scattering study of a small amide)138 and the

ΔE = 2.5 − 14.4/R

(10)

According to eq 10, a negative ΔE corresponding to a bound electronic state of the amide anion radical is achieved at R < 5.8 Å. As noted later, the experimental value of the amide electron affinity is affected by the 10−13−10−14 s lifetime of the state resonance, which results in a substantial uncertainty (Heisenberg broadening) for the binding energy. The revised value was estimated at 2−2.5 eV with a 1 eV uncertainty, and the range of R was extended to 12−25 Å.139 Another ion system was analyzed by Syrstad and Turecek who used time-dependent density functional theory (TDB3LYP/aug-cc-pTVZ) to calculate the electronic energies of ground and excited states of ion−molecule pairs consisting of neutral N-methylacetamide and methylammonium and guanidinium cations.135 The ions were placed at different distances and orientations with respect to the neutral amide. In both cases, excited electronic states were found that had most of the electron density in the amide π* orbital and were bound with respect to electron loss. The electron binding energy in the amide π* state was 2.6 eV in the A state of a contact, hydrogenbonded pair and then decreased to 1.55 eV in a higher (P) excited state when the ion and the amide molecule were at a distance of 7 Å. Analysis of the [CH3CONHCH3···+H3NCH3 + e−] system revealed bound amide π* electronic states at intermolecular distances extending up to 15 Å. Since the charged lysine ammonium, histidine imidazolium, and arginine guanidinium groups are internally solvated by hydrogen bonding to amide groups in peptide ions and therefore close to the peptide backbone, it appeared likely that the incoming electron can be stabilized in the amide π* orbitals in chargereduced peptide ions. Recently, computational studies of several charge-reduced peptide cation radical structures found zwitterionic structures with unpaired electrons in the amide π* orbitals as local energy minima on the potential energy surface.102,105 Figure 5 shows the molecular orbital of the

Figure 5. Structure and molecular orbital of the zwitterionic X state in the (AAVAR + 2H)+• cation radical from spin-unrestricted M06-2X/6311++G(2d,p) calculations. Atomic spin densities in black; atomic charges in purple italics. Data from ref 104.

ground (X) electronic state of (AAVAR + 2H)+• which illustrates the unpaired electron localization in the Ala1 amide group and the zwitterionic character of the cation radical.104 However, the electron structure of peptide cation radicals in the ground electronic state depends on the amino acid sequence, charge state, conformation, and also the computational method used, so general conclusions must be judged with caution.140 The mechanics of electron capture, transfer, and conversion of Rydberg to reactive valence electronic states have been 6698


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the equilibrium SS distance where the ν = 0 vibrational wave function had the highest density. Crossing from the 3s state was shifted off the equilibrium SS distance and required a vibrationally excited SS stretching mode, which is less populated than the ν = 0 state. Thus, the reason for more efficient transfer from the excited Rydberg was a more favorable coupling with the S−S σ* state.138,143 The role of Rydberg 3s and 4s states in mediating electron transfer was further examined for a series of related disulfide model ions, CH3SS(CH2)nNH3+ where the N···S distance was varied by increasing the number of spacer methylene groups (n = 1, 2, 3) in all-trans conformations. The authors concluded that the H12 decreased exponentially with the N···S internuclear distance but did so faster for the 3s Rydberg state.143 For another theoretical study of distance effects see ref 146. Related studies of a similar model system, CH3SSCH3···NH4+, indicated a more complicated picture involving not the ammonium 3s but 3p and 3d orbitals that mediated electron shuttling to the σ* orbital of the S−S group.144,145 The results of the theoretical ET studies were summarized in reviews as follows.147,148 In both ECD and ETD, the electron most likely enters a Rydberg orbital surrounding a positively charged site of the peptide ion. The accessible Rydberg orbitals are those with principal quantum numbers n = 3 and 4 for ET from a donor of a 0.6 eV electron binding energy and n = 3, 4, and 5 for capture of a free electron. The populations of the primary Rydberg states of different n were considered to be similar. The electron captured in such Rydberg orbitals can subsequently undergo fast electron transfer to any SS σ* or amide π*orbital that lies within a radial “shell” of average size ⟨r⟩ and thickness T that characterizes each Rydberg orbital (see below). At the same time, energy conservation requires that the receptor group electron binding energy exceed that of the donor Rydberg state. The rate of electron transfer to SS σ* or amide π*orbital must be faster than relaxation within the Rydberg orbital manifold. The last condition follows from the kinetic instability of ammonium 3s Rydberg states, which are known to undergo fast loss of H atoms,120−133 which would outcompete other dissociations. The difficulties with calculating accurate H12 coupling matrix elements led Simons and co-workers to develop an analytical model for estimating the distance dependence of intramolecular electron transfer from the ammonium Rydberg orbital, which was considered the primary attachment site, to the target S−S σ* or amide π* state.146,149 This model considers the spatial overlap between Rydberg ns (n = 3, 4, etc.) orbitals and the target valence orbital. Disulfide σ* or amide π* orbitals have a much smaller spatial extent than do 3s and higher Rydberg-type orbitals and thus are considered to be immersed in the Rydberg orbitals. Atom-like Rydberg orbitals have inner nodal surfaces and a radial probability density, P(r) = r2|Rn(r)|2, that acquires a maximum at r = n2a0/Z, where n is the principal quantum number, a0 is the Bohr unit of length, and Z is the atomic nuclear charge. An effective thickness (T) of the radial probability density was defined as a root-mean square of the radial expectation values (eq 13).149

studied in detail by Simons and co-workers.141−145 These studies mainly focused on σ* states of disulfide bonds but could be generalized to include amide π* states as well.141 Electron attachment to the disulfide group results in a fast cleavage of the S−S bond, which is one of typical dissociations in ExD of peptide ions containing disulfide bridges.113 Analysis of electron transfer rates from CH 3 − to the model CH3SSCH2CH2NH3+ cation, using electrostatic energy estimates and Landau−Zener theory, indicated that the ammonium charge site was 1−2 orders of magnitude more likely to receive the electron in a 3s Rydberg-like orbital compared to a direct attachment in the Coulomb-stabilized σ* orbital of the S−S group. An even greater difference (1−3 orders of magnitude) was estimated for capture of a free electron.134 Similar conclusions were drawn for electron transfer to the charged ammonium and neutral amide group in another small model ion, CH3CONHCH2CH2NH3+.141 The main reason for these differences were different coupling matrix elements (H12) between the orbitals on the anion donor and the receiving group. Although crossing to the SS σ* state of an estimated 2 eV binding energy (including Coulomb stabilization) can occur at longer distances than crossing to an ammonium 3s Rydberg orbital, the low probability for the state crossing due to its low H12 coupling term (eq 11) more than offsets the longer distance. PLZ = 1 − e−2πH12

2

/(ℏvrel |F12|)

≈

2πH12 2 ℏvrel|F12|

(11)

The H12 coupling terms can be geometrically interpreted as depending on the spatial overlap of the interacting orbitals. The SS σ* orbital, despite its higher energy, is more compact than the ammonium 3s Rydberg and therefore shows less overlap with the donor orbital at distances corresponding to the surface crossing. Simons et al. also considered competitive electron transfer to the SS σ* orbital and ammonium ground (3s) and excited (presumably 4s) state.138 The calculated ET probabilities showed large variations (>10-fold) with the anion electron binding energy that affects the electron transfer exothermicity (ΔE). The overall conclusion was that the probability for electron transfer was small (20 Å distance from the peptide backbone, so it covered regions of radial density of Rydberg states up to n = 6. The potential bias steers the free electron toward the positive end of the dipole and lowers the energies of Rydberg states with radial density in that region.102 This favors further electron transfer to the valence π* orbitals of the group situated at the positive end of the dipole. Electron capture in the histidine residue can trigger rearrangements and loss of H atoms but does not promote backbone cleavages.102 The peptide ion structure that gave rise to the substantial dipole moment was confirmed by ion mobility and action spectroscopy.156 In contrast, electron transfer from atomic or molecular donors, for example, cesium or fluoranthene anion radical, is less susceptible to the ion dipolar field. In ECID, the ion−atom encounter occurs at random orientations and lasts only a few femtoseconds (eq 4) during which the electron donor and acceptor cannot reorient to achieve the energetically most favorable ion−dipole configuration. In ETD, the cation− anion pair is engaged in an orbiting complex where the ions conserve their angular momentum, which prevents them from being aligned by weak ion−dipole or ion-induced dipole forces. Moreover, ET from the anion depends more strongly on the reaction exothermicity and H12 coupling elements and thus appears to be less sensitive to the peptide cation dipole moment. Dipolar effects were also used to interpret differences in the ECD, ETD, and ECID spectra of several phosphopeptides that also had substantial dipole moments.105 Dipole effects on backbone dissociations following electron attachment were further elaborated by Simons and coworkers.139 These authors considered their previous model of Coulomb stabilization (C) for electron attachment to the SS σ* and amide π* states in doubly protonated (N-Ac-Cys-(Ala)jLys)2 (j = 10, 15, and 20).150 Assuming protonation at the lysine ε-amino groups and a fully extended conformation of the peptide dication, the distance between the SS and charged groups for n = 15 was estimated at 24 Å, resulting in a Coulomb stabilization of C = 2 × 14.4/24 = 1.2 eV. Likewise, the Coulomb stabilization of the farthermost alanine amide groups that undergo bond cleavage was estimated at 2.2 eV. Both values were barely sufficient to allow for a direct electron attachment to these groups but made transfer from 3s and 4s ammonium Rydbergs impossible on energy grounds. This created a puzzling situation because the previous analysis of simple models indicated that Rydberg-like orbitals of low principal quantum numbers (n = 3−5) were most efficient for electron transfer. One might note that one of the arguments for the need for very fast intramolecular ET from ammonium 3s Rydbergs was that the estimated ET rates to the peptide valence states must exceed those for the dissociation by loss of H atoms, which is also fast in ammonium radicals. However, ammonium radicals

where q was NH3+ or (CH3)3N+CH2CH2CH2CO and R was H or CH3. ETD of these peptide ions showed disulfide bond cleavages regardless of the type of the charged group. In particular, peptide ions with nonprotonated charged groups gave somewhat higher yields of disulfide bond cleavage products than did the nonderivatized ions containing multiple charged ammonium groups. These results showed that the disulfide group cleavage was not conditioned upon the presence of protons or hydrogen atoms formed by charge reduction, contrary to earlier suggestions.113 This was consistent with the general assumptions of the Simons’ model and also with studies of hydrogen atom adducts to disulfides that found competitive cleavages of S−C bonds,152 which are less frequent in ExD (see section 6.3).153,154 Gunawardena et al. estimated the Coulombassisted electron binding energy of the SS group as 2.3 eV, which was lower than those of both the ammonium and trimethylammonium groups (4−5 eV for 3s states).151 Hence, the ammonium 3s Rydbergs could not shuttle electrons to the SS σ* orbitals on energy grounds. Other applications of simple distance rules to ETD of conformationally folded peptide ions were found to be less straightforward. For example, ETD spectra of doubly charged phosphopeptides, (Ala-pSer-Ala-Ala-Arg + 2H)2+, (Ala-AlapSer-Ala-Arg + 2H)2+, and (Ala-Ala-Ala-pSer-Arg + 2H)2+, with well-defined conformations105 showed backbone cleavages next to the N-terminal alanine residue despite the fact that the amide groups and dissociating bonds were in the “forbidden” zone of the ammonium Rydberg orbitals with low electron transfer probabilities from the 3s−6s states. In contrast to the fully extended peptide model150 that was used to test the simple distance rules, most peptide ions studied so far have shown folded structures in which many or nearly all amide groups were situated within the ET zones of the charged groups. It appears that the simple analytical model is more useful as a general concept rather than a practical predictor of backbone dissociation frequencies in peptide ions. A similar conclusion was reached recently in a study that used a modified formula for molecular Rydberg orbitals that included the so-called quantum defect that shrinks the orbital radial extent by 40−60%.155 6700


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dissociations have been studied in detail.132 Loss of H atoms from simple ammonium radicals is slightly exothermic, faces very low potential energy barriers (90% D content) conversion of exchangeable protons for deuterons in gas-phase peptide ions is difficult to achieve and requires special precautions to suppress back-exchange in the electrospray ion source or ion trap.97 For example, solution H/D exchange in (Ala-Arg + 2H)2+ ions with 10 exchangeable protons achieved a maximum of D8 species in the electrospray mass spectrum with relative intensities that were not seriously affected by 13C and 15 N isotopologue overlaps. ECID showed loss of H and D in a 3.3:1 ratio, which greatly favored loss of H over the expected statistical 1:4 ratio based on the loss of exchangeable hydrogen atoms only.97 The authors interpreted the H/D loss data by large kinetic isotope effects but could not exclude loss of H atoms from nonexchangeable positions. Loss of H/D in ECID of the D7 isotopologue of (Gly-Lys + 2H)2+ (total of eight exchangeable protons) gave a H/D loss ratio of 1:2, again exceeding the 1:8 ratio predicted statistically.158 Large isotope effects on H/D loss are common in dissociations of ammonium radicals.121,126,129,132

in n = 4 and higher Rydberg states are strongly bound with respect to H atom loss,135 so the transfer rate issue is less about dissociation and more about competition between conversion to the dissociative 3s and valence SS σ* and amide π* states. If the transfer rate to the valence states is always greater than that to the lower Rydberg states, then the absolute rates are unimportant, because they exceed the dissociation rates by several orders of magnitude. To resolve the energy conservation puzzle, Simons and co-workers analyzed stabilization by ion dipole effects (D) of the SS σ* and amide π* states in a helical peptide conformation and concluded that the dipole effects were an important contributor to stabilization.139

6. DISSOCIATIONS OF HYDROGEN-RICH PEPTIDE RADICALS AND CATION RADICALS Electron attachment to peptide cations greatly affects the reactivity of charge-reduced peptide radicals and cation radicals and triggers radical reactions. The typical dissociations observed in ExD are sketched in Figure 6 and include loss of

Figure 6. Typical dissociations of hydrogen-rich peptide cation radicals upon ExD.

H atoms, ammonia, and side-chain groups, as well as disulfide bond cleavages and backbone dissociations of N−Cα bonds. The primary products of these competitive fragmentations then can undergo consecutive reactions that occur spontaneously in ECD or can be promoted by supplemental ion activation. In addition to dissociations, which are directly observable in the mass spectra, peptide radicals and cation radicals have been reported to undergo rearrangements forming isomers. Investigations of rearranged peptide radicals and cation radicals required special techniques, as described in Section 6.7. 6.1. Loss of H Atoms

Loss of a hydrogen atom is a common dissociation of peptide radicals and cation radicals upon ECD and ECID and often produces major even-electron fragment ions that correspond to the lower protonation state of the precursor ion. Mechanistic investigations of these dissociations have so far relied on analogies from studies of smaller systems containing ammonium,120−132 pyrrolidinium,133 guanidinium,157 and imidazolium108 groups that mimic the respective charged groups in lysine, proline, arginine, and histidine residues. These groups show major losses of hydrogen atoms upon reduction by electron transfer, and the kinetics of these 6701


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ExD was reported to cause little intramolecular H/D exchange in charge-reduced peptide cation radicals or their dissociation products,161−164 contrasting other modes of activation that rely on proton migration and effectively scramble the exchangeable hydrogen atoms. ECD and ExD studies using D-labeling in nonexchangeable positions are rare.165 O’Connor et al. reported the ECD spectrum of a doubly charged hexadecapeptide ion, Arg-Ala-Ala-Ala-Gly*-AlaAsp-Gly*-Asp-Gly*-Ala-Gly*-Ala-Asp-Ala-Arg, in which the αpositions of all glycine residues (G*) were labeled, so the ion contained eight deuterium atoms in nonexchangeable positions and 31 exchangeable protons. The ECD spectrum showed an abundant loss of light H but no loss of D, indicating that the glycine methylene groups were not involved.165 Studies of H loss in ETD using anion electron donors are ambiguous because ET competes with proton transfer,166 which leads to even-electron ions that have identical m/z as the products of H-loss following charge reduction, and thus these two processes are difficult to distinguish.

Scheme 4

6.2. Loss of Ammonia

Loss of ammonia is a very common dissociation of peptide cation radicals produced by electron capture or transfer. 15N labeling of the N-terminal amino group in small lysineterminated peptide ions, (Ala-Lys + 2H)2+, (Lys-Lys + 2H)2+, (Ala-Ala-Lys + 2H)2+, and (Gly-His-Lys + 2H)2+, has established that the ammonia molecule originated with high selectivity (>95%) from the N-terminal ammonium group.167 Indirect evidence also followed from the ETD spectrum of Nterminal acetylated peptide ion (N-Ac-Ser-Asp-Lys-Pro-AspMet-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys + 3H)3+ that did not show a detectable loss of NH3 despite the presence of three lysine ε-NH3 groups.168 In contrast, ECID of peptide ions Cterminated with arginine showed loss of ammonia from the guanidinium group. This was determined indirectly for (ProArg + 2H)2+ and (Cys-Arg + 2H)2+, which both showed loss of NH3 from the charge-reduced cation radicals.159 While the argument for (Pro-Arg + 2H)+• was obvious because the ion had no N-terminal ammonium group, that for (Cys-Arg + 2H)+• was subtler. Loss of the N-terminal ammonia from cysteine-terminated peptides produced cation radical fragments that underwent extremely fast loss of the side-chain SH radical, so as shown for (Cys-Lys + 2H)+• (Scheme 4), the intermediate ion at m/z 234 was absent in the ECID mass spectrum. In contrast, the dissociation of (Cys-Arg + 2H)+• did give a primary fragment ion (m/z 262, Scheme 4), which was attributed to an isomer formed by loss of ammonia from the guanidinium group.159 Mechanistic details for the loss of ammonia from guanidinium radicals are limited. The radical formed transiently by electron transfer to protonated arginine amide showed a loss of ammonia that presumably originated from the guanidinium radical group.95 Elimination of ammonia from the prototypical guanidinium radical, •C(NH3)3, has a low threshold energy (ΔHrxn = 60 kJ mol−1) but requires >158 kJ mol−1 in the transition state and presumably proceeds on the potential energy surface of an excited electronic state.157 The energetics and dynamics of loss of ammonia from Nterminal positions in peptide radicals and cation radicals have been studied for (Gly-Gly-NH2 + H)• (ref 169), (Gly-Lys + 2H)+• (ref 101), and (Ala-Arg + 2H)+• (ref 103). The (GlyGly-NH2 + H)• radical requires only 9 kJ mol−1 in the transition state for loss of the N-terminal ammonia. The

dissociation becomes spontaneous upon rotation of the H3N group about the CH2CO bond away from the coplanar conformation with the amide carbonyl. The elimination is highly exothermic (ΔHrxn = −129 kJ mol−1) and involves a hydrogen-bonded noncovalent complex (Scheme 5).169 Elimination of the N-terminal NH3 group from (Gly-Lys + 2H)+• is extremely facile, requiring only 3 kJ mol−1 in the transition state (TS), and proceeds exothermically with ΔHrxn = −181 kJ mol−1.101 The competing loss of the lysine ε-NH3 group is also exothermic (ΔHrxn = −112 kJ mol−1) but requires a somewhat higher TS energy (27 kJ mol−1). These data indicate that the preferential elimination of the N-terminal ammonia is a kinetic rather than thermodynamic effect. The dynamics of ammonia elimination from the N-terminal position was studied for (Ala-Arg + 2H)+•.103 The elimination is extremely facile, proceeds on the potential energy surface of 6702


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Loss of ammonia was also common in ETD of pentapeptide ions of the (AAXAR + 2H)2+ type (X = Ala, Val, Leu, Ser, Asp, Asn, His, Lys, and Met) where it amounted to 30−50% of the total fragment ion intensity. The origin of the eliminated ammonia molecule was not established, and the variations in the fragment ion relative intensities were not readily related to the nature of the amino acid residues in these peptides.104

Scheme 5

6.3. Disulfide Bond Cleavage

Electron attachment to multiply charged peptide and protein cations containing disulfide groups results in a facile cleavage of the S−S bond. An early paper on ECD of large multiply charged peptide ions reported preferential S−S bond cleavage, whereby the even-electron RSH fragment ion was formed preferentially from the peptide part (B) carrying more basic residues and presumably a higher charge (eq 14).113 (mH+)A‐SS‐B(nH+) + e− → ASm +• + BSH(n − 1) +

the ground electronic state, and competes with the ammonium proton migration onto the amide oxygen (Figure 7). Electron

for

n>m

(14)

For example, the ECD spectrum of an (ASSB + 10H)10+ precursor ion, where A and B were 38- and 48-residue long peptides containing 6 and 7 basic residues (arginine, lysine, and histidine), respectively, gave a better fit for the stable isotope envelope of BSH4+ than for that of BS4+• and likewise for AS5+• and ASH5+. This preference was explained by polarization of the S−S bond in the dissociation of the putative RS(H)•SR′ radical transiently formed by hydrogen atom attachment to the disulfide bond.113 A S−S bond cleavage was also facile upon ET from SO2−• to cysteine-linked peptide ions (Asn-Cys-Pro-Arg)(Cys-Phe-IleArg) and (Cys-Glu-Val-Phe-Arg)(Leu-Asp-Gln-Trp-Cys-GluLys-Leu), denoted peptides I and III, respectively.70 The ETD spectra of triply protonated peptide I ions showed similar relative intensities of A- and B-chain thiyl (A,B−S•)+ and thiol (A,B−SH)+ fragments, indicating no preference for H atom attachment to the sulfur atoms. ETD of peptide III ions showed dramatic effects depending on the charge state of the precursor ions. Whereas the doubly charged precursor ion showed charge reduction but very little fragmentation, ETD of the triply and quadruply charged precursor ions showed dominant S−S bond dissociation accompanied by backbone cleavages.70 The hydrogen atom capture mechanism proposed in the early work was based on preliminary calculations of the H-atom affinity of the disulfide group.113 The difficulty with the hydrogen atom capture mechanism was that the S−S group is less basic than most polar groups in the peptide ion. The proton affinity of the S−S group in CH3SSCH3 (PA = 815 kJ mol−1 from the NIST database170 and 806 kJ mol−1 from highlevel ab initio calculations152) is >100 kJ mol−1, lower than the proton affinities of amino acids.171,172 Hence, the S−S group is not likely to be protonated or even capable of internally solvating the charged groups in peptide ions to bring protons to its vicinity and thus facilitate hydrogen atom transfer after charge reduction.173 Another study examined model disulfide radicals, CH3S(H)•SCH3 and (dithiolane + H)• that were generated by femtosecond electron transfer to gas-phase cations152 or by dissociative recombination in an ion storage ring.116 The radical intermediates were calculated to reside in shallow potential energy wells and found to undergo competitive cleavages of the S−S and C−S bonds. This contrasted ECD spectra of peptide ions that were reported not to show products of C−S bond cleavages.113 Furthermore, the 98 kJ mol−1 exothermic H atom addition to the S−S group had

Figure 7. Competing reaction pathways on the potential energy surface of the ground electronic state of (Ala-Arg + 2H)+•. Data from ref 103.

attachment to the (Ala-Arg + 2H)2+ ion leads to a dissociative potential energy surface of the ground electronic state of the cation radical. At a low vibrational excitation (ν = 0), the system follows the gradient along the internal coordinate corresponding to the ammonium N−H stretch and rapidly proceeds to an exothermic proton migration to the amide carbonyl, forming an aminoketyl radical. The time period for this motion was calculated to be about 70 fs, so the isomerization was predicted to be very fast, and the kinetic energy was mainly contained within the aminoketyl group. At higher vibrational excitation of the precursor ion, the torsional motion about the CαCO bond in the alanine residue switched the system into following another reaction coordinate, which was the CαNH3 stretch that had a steeply dissociative profile. The departing ammonia molecule reached a point of no return within 100 fs after electron attachment and continued to fly away past the shallow energy minimum of the noncovalent ion−NH3 complex. The experimental branching ratio for the loss of ammonia and proton migration, following collisional electron transfer from Cs atoms, favored the former dissociation by 3.6:1.97 6703


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to overcome a small potential energy barrier that depended on the conformation in the transition state.152 These results indicated that there was no long-range attractive potential for the hydrogen atom to zoom in onto a distant S−S group and raised some doubt about the role of hydrogen atom adducts in the selective disulfide bond cleavages in ExD. The H-atom capture mechanism was further tested by two experimental studies that used model peptides with extended conformations and fixed charges, as discussed in section 4. Efficient cleavage of the S−S bond was observed in ETD of peptide ions that lacked hydrogen atom donor groups151 and in ECD of peptide ions in which the charged groups were 24 Å away from the S−S group. Both results were incompatible with the H-atom capture mechanism. A recent study by O’Connor and co-workers dealt with ECD of peptide ions consisting of Lys-Met-Gly-Ile-His-Ala-Cys(S,Se)-Val-Glu-Phe-Lys residues that were linked by S−S, S− Se, and Se−Se bonds.153 In all cases, the predominant dissociation of charge-reduced cation radicals was cleavage of the S−S, S−Se, or Se−Se bond yielding complementary pairs of thiyl (S•), selenyl (Se•), thiol (SH), and selenol (SeH) fragment ions. Interestingly, the dissociations of S−Se linked peptide ions showed (S•)/(SH) intensity ratios of ∼0.1, while the (Se•)/(SeH) ratios were ≫1. The authors discussed the results in terms of different electron affinities of the disulfide and diselenide groups, but noted that the energy data did not allow a consistent interpretation of results for different peptide chains. One might note that the S−H bond dissociation energy is much greater than that for the Se−H bond,174 and thiyl radicals are known to abstract hydrogen atoms from Cα positions in amino acids and peptides.175 The preference for thiol fragment ions thus can be due to postcleavage hydrogen atom transfer from the Se-containing fragment. An interesting observation made by O’Connor and co-workers was that ECD also produced minor fragment ions due to cleavages of the C−S and C−Se bonds.153 Further insight has been gained from ETD studies dealing with peptides containing intramolecular S−S bonds that demonstrated the competitive nature of S−S and N−Cα bond dissociations.154,176,177 S−S bond cleavage upon electron attachment according to the Simons mechanism forms paired thiol radical and thiolate anion. These can be readily quenched by hydrogen atom transfer to the thiyl radical and proton transfer to the thiolate anion, forming a dithiol isomer of the charge-reduced disulfide. Both the H-atom and proton transfers are energetically possible. Transfers of H atoms from peptide Cα positions to cysteyl S-radicals have been studied by experiment and theory.178−180 The basicity of the thiolate group (1640−1650 kJ mol−1)181 vastly exceeds those of typical basic groups in peptides to allow exothermic proton transfer. Consistent with these rearrangements, the ETD mass spectra of several peptides with internal S−S bonds displayed intense charge-reduced peptide cation radicals that did not dissociate under the experimental conditions (Scheme 6). In addition to nondissociating charge-reduced ions, the ETD mass spectra displayed backbone c- and z-type fragments that must have included cleavages of at least two bonds, one S−S and the other N−Cα, in the charge-reduced ion. Xia and co-workers discussed two alternative mechanisms for these dissociations.154 One involved a radical attack by the backbone Cα-radical at the S−S group to form a cyclic sulfide in the C-terminal z fragment ion (Scheme 6). This mechanism was supported by the MS3 spectra of cysteine-containing z ions, for example, z5-Phe-

Scheme 6

Leu-Lys-Lys-Cys, that showed amino acid residue scrambling and loss of lysine residues, consistent with a cyclic structure. Cradical attack at sulfide atoms requires relatively low activation energies (50−90 kJ mol−1),182,183 which are within the range of activation energies of competing dissociations of ExD fragments. C-radical attack on the S−S group is known to proceed readily and with low activation barriers, as studied by experiment12 and theory.184 The alternative mechanism considered by Xia and co-workers involved Cα-H transfer to the sulfhydryl radical, followed by backbone cleavage of the peptide Cα radical.153 This mechanism was found to be less likely, because it would form c − H fragment ions that were not observed in the spectra. Xia et al. also reported that ETD produced fragment ions resulting from C−S bond dissociations,153 consistent with the previous studies of disulfide radicals.116,152,173 6.4. Peptide Backbone N−Cα Bond Cleavage

Backbone dissociations providing information on amino acid sequence and location of post-translational modifications such as phosphorylation, glycosylation, and others are the most important reactions of hydrogen-rich peptide cation radicals.185−202 The specific feature of ExD techniques is that the predominant backbone cleavages break the bonds between the amide nitrogen atoms and the Cα atoms of the neighboring amino acid residues, or N−Cα bond cleavage for short. The dissociation is accompanied by hydrogen transfer to the amide group, producing N-terminal fragments that are even-electron species (c-type ions), and C-terminal fragments that are hydrogen-deficient radicals (z-type ions). Depending on the charge state of the peptide precursor ion and the relative basicity of the N-terminal and C-terminal fragments, the dissociation can give rise to c-type ions, z-type ions, or both. If the primary fragments gain sufficient internal energy, they can 6704


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Scheme 7

the Cα−CO or CO−NH bonds leading to a-, b-, x-, and y-type fragment ions (Scheme 7). The a and y ions are observed only as minor products in ECD209,221 and even more rarely in ETD. An experimental study found that hydrogen atoms generated in a discharge source neither formed adducts with nor triggered dissociation of singly charged desR1-bradykinin peptide ions trapped in an ion-cyclotron resonance cell under conditions similar to those employed in ECD.222 D-atoms reacted slowly with desR1-bradykinin ions over 90 s, showing ∼10% H/D exchange and indicating D atom addition followed by H atom elimination, but no backbone cleavage.222 The hot-H atom hypothesis was further tested by statistical169 and Born− Oppenheimer dynamics calculations in amide models.223 Application of Keizer’s steady-state theory224 indicated that the rate constant for H-atom addition would increase to 10−12 molecules cm−3 s−1 for translationally hot hydrogen atoms with >1 eV kinetic energy. However, to achieve reaction rates that would be competitive with other intramolecular processes, the reaction volume for the H-atom recapture would have to be reduced to 10−20−10−22 cm3, so only hot hydrogen atoms originating in a close proximity of the amide group could be captured.169 Uggerud and co-workers carried out Born− Oppenheimer dynamics calculations for hot H atom addition to small neutral amide molecules. The trajectory calculations indicated that the majority of H atoms would be ejected from the short-lived transient adducts and only about 3% of those would proceed to N−Cα bond dissociation.223 The H-atom expulsion−recapture reaction was found to poorly compete with another process involving ammonium and amide groups in peptide radicals and cation radicals,169 which was identified as proton-coupled electron transfer (PCET).225,226 Analysis of electron density distribution in transition states indicated that intramolecular hydrogen atom transfer from an ammonium radical to a proximate amide group proceeded as an asynchronous migration of an electron−proton pair, whereby fast electron migration was followed by slower proton migration. Density functional theory calculations indicated that the spin density, which was localized in the ammonium group of the reactant, mostly shifted to the amide carbonyl in the transition state for the proton N···H → OC migration.169 The isomerization of the ammonium radical to the aminoketyl radical intermediate was exothermic and required only a small energy barrier in the transition state.169 This led to a reformulation of the so-called Cornell model to include

undergo consecutive dissociations. This is more common in ECD, where consecutive dissociations can result in side-chain loses203−210 or ring cleavages211 or form smaller backbone fragment ions of the a-, x-, or z-type. The studies of backbone dissociations on ExD have followed two main lines. One type was survey studies focused on the effect of amino acid residues on the frequency of backbone cleavages.212−216 The other type was mechanistic studies that focused on the dissociation energetics, kinetics, or specific modifications affecting the peptide cation radical valence or electronic structure. 6.4.1. Mechanisms of N−Cα Bond Cleavage. The mechanism of N−Cα bond cleavage has been discussed from the very beginning, starting with the first paper on ECD,55 and the early discussions were reviewed by Zubarev and co-workers in 200256 and 2003.57 The first mechanism, proposed by McLafferty and co-workers,55 considered electron attachment to a protonated backbone amide group to form an aminoketyl radical intermediate that underwent a β-fission involving the N−Cα bond on the C-terminal side of the affected amide group. However, since amide groups are less basic than the side-chain groups at lysine, arginine, and histidine residues, extensive amide protonation in multiply charged gas-phase ions was unlikely. The next mechanistic variation proposed by the Cornell group considered electron attachment to a chargecarrying group in the basic residues (lysine, arginine, or histidine) forming an unstable radical intermediate. That was presumed to dissociate, producing a kinetically hot hydrogen atom that would be captured by an amide group forming again an aminoketyl radical intermediate and proceeding to N−Cα bond dissociation.54,113,217,218 In addition, another idea was floated by the authors and attributed to J. I. Brauman in a footnote113 that the N−Cα bond cleavage may proceed from a repulsive σ* state, which is accessed from a high Rydberg state formed by electron capture. This corresponds to the traditional description of dissociative electron recombination of small ions.135 The hot hydrogen atom hypothesis was subsequently scrutinized by experiment and quantum chemistry calculations. Addition of a H atom to amide carbonyl oxygen atoms, albeit exothermic,115,219,220 requires overcoming potential energy barriers of 38−68 kJ mol−1 that would make a thermal reaction very slow, with rate constants calculated at k ≈ 10−18 molecules cm−3 s−1.169 A comparable energy barrier was found for H-atom addition to the amide carbonyl carbon atom forming a transient oxygen-based radical.219 A β-fission in the latter would break 6705


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Scheme 8

Figure 8. ECD mass spectrum of (Ac-Kbt-Gly5-Kbt-NH2)2+ showing backbone N−Cα bond dissociations in the Gly5 segment. Adapted with author’s permission from ref 227.

PCET instead of hydrogen atom loss−recapture in the isomerization step (Scheme 8).95 The general validity of the Cornell mechanism has been challenged by several experimental studies. A communication by Hudgins, Marshall, and co-workers reported the ECD mass spectrum of a modified peptide ion (Ac-Kbt-Gly5-Kbt-NH2)2+ in which the charge was localized in quaternary ammonium groups and the N-terminal ammonium and C-terminal carboxyl protons were blocked by N-acetylation and amidation (Figure 8).227 The spectrum demonstrated that backbone N−Cα bond dissociations occurred even in the absence of ammonium protons. In another series of studies, electron attachment to protonated arginine95,97 and histidine residues96,228 was found to form the respective radical guanidinium and imidazolium intermediates, which were good proton or hydrogen atom acceptors but poor hydrogen atom donors and, hence, they were unlikely to engage as group X in the Scheme 8 mechanism. Despite this reactivity trend, peptide ions protonated at arginine residues do undergo abundant backbone N−Cα bond cleavages upon electron capture.213,214 It might be noted that the majority of peptides, namely, those from protein digests, contain ammonium groups at the N-terminus and in the C-terminal lysine residue that can undergo PCET upon electron attachment and produce aminoketyl intermediates for N−Cα bond dissociations in keeping with the Cornell model. However, it is difficult to explain the incidence of several N−Cα

bond cleavages along the peptide backbone as being triggered by reduction of a single ammonium group. Applications of the Cornell model to explain the dissociations of peptide cation radicals are hampered by the incomplete knowledge of the gas-phase peptide ion structure, namely protonation sites and conformation. For example, ECD of the frequently studied doubly protonated ion of the neuropeptide substance P (Arg-Pro-Lys-Pro-Gln-Gln-PhePhe-Gly-Leu-Met-NH2) showed a series of c4 through c10 sequence fragment ions by dissociations of all N−Cα bonds from Pro4 to the C-terminus.229 The relative intensities of the c fragment ions showed some variations in different survey-like studies.230−232 When the precursor ion was cooled from ambient temperature (308 K) to 86 K, the ECD spectrum, recorded at a substantially reduced signal-to-noise ratio, showed only two cleavages at the Phe and Leu residues giving rise to c7 and c10 fragment ions, respectively. The temperature effect was interpreted by conformational effects.229 The gas-phase ion was presumed to exist as multiple conformers at 308 K, thus allowing access of the Lys ammonium group to various amide groups along the backbone. The authors assumed that that upon cooling, the conformational diversity was reduced to conformers in which the Lys ammonium had access mainly to the Phe and Leu amide groups. Interestingly, this analysis was based on an ion mobility study of Gill et al. that also reported force-field molecular dynamics (MD) of the substance P dication.233 According to Gill et al., the Lys NH3 group had 6706


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Scheme 9

benzylammonium radicals formed by collisional electron transfer could be explained by the electron entering the π* electronic system of the aromatic ring containing electronwithdrawing substituents to form zwitterionic intermediates.128 This interpretation was inspired by intramolecular electron transfer studies of Closs and co-workers.237−239 The 2003 study by Simons and co-workers (ref 150, see above) posited that electron attachment to the σ* state of a remote S−S group can be facilitated by Coulomb stabilization by a remote charged group. The UW model of backbone dissociations postulates that the electron enters a Coulomb-stabilized π* state of an amide group in the charge-reduced peptide ion to form a zwitterionic intermediate comprising an enol−imidate anion radical and the original cationic sites. These intermediates were thought to correspond to excited electronic states of the transient charge-reduced species, based on electron structure calculations of small model systems.135 However, analysis by density functional theory of several penta- and heptapeptide cation radicals revealed that zwitterionic intermediates resided in local energy minima on the potential energy surface of the ground or low excited states of peptide cation radicals (cf. Figure 5). Excited electronic states of hydrogen-rich peptide cation radicals form a dense manifold of several (>10) states fitting within a 1 eV energy interval above the ground state. This is consistent with the narrow range of recombination energies for various functional groups in peptide ions (Table 1).

very similar probability of being near any of the Gln5 through Met11 amide carbonyls, and the MD calculations indicated that the average number of internally solvated carbonyls increased from 2.6 to four in the lowest free-energy structures.233 Thus, if the ion population at 86 K was reduced to the lowest freeenergy conformers, one should expect at least four backbone cleavages rather than just two. Conformational effects on backbone dissociations of charge-reduced peptide and protein ions were discussed in several other studies.234−236 ETD of conformationally well-characterized pentapeptide ions, such as (Ala-Ala-His-Ala-Leu + 2H)+(ref.156),showed dissociations of all four N−Cα bonds upon electron transfer from neutral or anion donors.102 The lowest-free energy conformer, which was dominantly represented in the ion population,156 showed an intramolecular hydrogen bond between the N-terminal ammonium and the Ala4 amide carbonyl; yet the most abundant fragment ion was c3 by backbone cleavage at the His3 residue. The difficulties with the Cornell mechanism led to a conceptually different model that was independently introduced by research groups at University of Utah and Washington and has been dubbed the Utah−Washington (UW) model.95 The UW model builds on previous suggestions for electron attachment to gas-phase ions that affected functional groups remote from the charge carrying sites.128,150 A 1996 study concluded that some unusual dissociations of 6707


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Scheme 10

fragments formed by N−Cα bond cleavage form a complex169 in which they can undergo various ion−molecule reactions (see below). For example, the c/z ion−molecule complex from (Gly-Phe-NH2 + 2H)+• was found to have an extremely low barrier for catalyzed interfragment proton transfer that isomerized the enolimine tautomer of the c fragment into the more stable amide form.244 Analogous catalyzed prototropic isomerizations in ion−molecule complexes have been reported for a large number of fragment ions.245−258 The competitive prototropic isomerizations and N−Cα bond cleavages in peptide cation radicals have been studied computationally for a few systems.102,104 (Ala-Ala-His-Ala-Leu + 2H)+• was found to be a zwitterion in the ground electronic state that had the unpaired electron in the π* orbital of the Ala1 amide group. Scheme 10 shows the transition-state structures and energies (in kJ mol−1) for the competitive isomerization by proton migration forming an aminoketyl intermediate and direct N−Cα bond dissociation forming an ion−molecule complex.102 Proton migration in the zwitterion had a very low TS energy (TS1) for exothermic isomerization to the aminoketyl intermediate, which can dissociate through TS2 to form an ion−molecule complex (complex 1). This pathway corresponds to the Washington variant of the UW model. The Utah variant involves TS3 for the direct N−Cα bond dissociation in the zwitterion, forming another complex (complex 2). The complexes differ in the structure of the neutral c1 fragments, which are the enolimine (complex 1) and zwitterionic enolimidate (complex 2) tautomers of alanine amide. Complex 2 can readily isomerize to complex 1 or further to alanine amide. It was argued on the basis of the calculated TS energies and RRKM rate constants that very fast isomerization through TS1 converts the cation radical to the aminoketyl intermediate, which then dissociates through TS2. A reversible isomerization to the cation radical and dissociation through TS3 was substantially slower due to both the higher TS3 energy and depletion of the cation radical reactant.102 Very similar relative energies for transition states, intermediates, and ion−molecule complexes were reported for isomerizations and dissociations of zwitterionic (Ala-Ala-Val-Ala-Arg + 2H)+• cation radicals (Figure 5) where the N−Cα bond dissociation in the aminoketyl radical intermediate had a lower TS energy

The excited states consist of Rydberg as well as amide and sidechain π* states that differ in the electron density distribution and can trigger dissociations through different reaction channels.101−103 Starting with the initial electronic state or an intermediate having the electron in the amide π* system, the UW model presents some variants for further reaction steps (Scheme 9). The Washington variant (right-hand branch in Scheme 9) emphasizes the high basicity of the enol−imidate anion radical and posits that the anion is quenched by proton transfer from a charged or neutral site to form an aminoketyl radical, which then undergoes N−Cα bond cleavage analogous to that considered by the Cornell model.55,240,241 The Utah variant (left-hand branch in Scheme 9) emphasizes the radical nature of the enol−imidate anion radical and posits that the carbonylcentered radical promotes β-fission of the N−Cα bond at the Cterminal side of the amide group carrying the reducing electron. This forms the zk fragment ion and a complementary cl fragment, which is deprotonated in the amide group. Formation of the regular cl fragment ion necessitates proton transfer to the highly basic enol−imidate anion. Yet another alternative mechanism has been proposed recently that considered βfission of the N−Cα bond at the N-terminal side of the amide radical group to form the zk+1 cation radical and cl−1 enol− imidate fragments.242 Distinguishing between the variants of the UW model is difficult because it requires some subtle experiments and high-quality theoretical calculations. The Washington and Utah variants differ in that the former leads to the formation of an enolimine tautomer of the c fragment ion, whereas the latter can produce the more stable amide tautomer by intramolecular protonation of the enol−imidate intermediate. Infrared multiphoton photodissociation (IRMPD) action spectroscopy was used to investigate the structure of the c1 fragment ion from ECD of the Gly-Lys dipeptide that was tagged at the N-terminus with the fixedcharge tris-(trimethoxyphenyl)phosphonium (TMPP) group.243 The IRMPD spectrum showed a better fit to the calculated IR spectrum of the amide tautomer, implying that the stable, long-lived ion was an amide. The implications for the N−Cα cleavage mechanism of this experimental result must be interpreted with caution. The complementary c and z 6708


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Table 3. Transition State Energies for N−Cα Bond Dissociations in Aminoketyl Radicals energya b

species

BDE

CH3C (OH)NH−CH3 H2NCH2CH2C•(OH)NH−CH3 CH3C•(OH)NH−CH2CH2NH2 CH3C•(OH)NH−CH2CH2NH3+ CH3C•(OH)NH−CαH-Lys H2NCH2C•(OH)NH−CH2CONH2 H3N+CH2C•(OH)NH−CH2CONH2 H2NCH2C•(OH)NH−Cα(H)-ArgH+ Pro-C•(OH)NH−CH2COOH Gly-Pro-C•(OH)2 H2NCH2C•(OH)NH−Cα(H)Pro H2NCH2C•(OH)NH−Cα(H)Lys(H+) (H+)LysCONH−Cα(H)C•(OH)2Lys (H+)GlyNH−Cα•(H)(OH)NH2(CH2Ph) AlaC•(OH)NH−Cα(H)Ala-His(H+)-Ala-Leu Ala-AlaC•(OH)NH−Cα(H)His(H+)-Ala-Leu Ala-Ala-His(H+)C•(OH)NH−Cα(H)Ala-Leu Ala-Ala-His(H+)-AlaC•(OH)NH−Cα(H)Leu AlaC•(OH)NH−Cα(H)Ala-Val-Ala-Arg(H+) Ala-Ala-HisC•(OH)NH−Cα(H)Ala-Arg(H+) AlaC•(OH)NH−Cα(H)Ala-His-Ala-Arg(H+) (H+)Ala-Ala-β-Ab(C•(OH)NH−Cα(H)-Ala-Alaf (H+)Ala-AlaC•(OH)NH−Cα(H2)β-Ab-Ala-Alaf

11 23 38 76b −12 −1 (9)c 67b 7−24b,c 18 −89d −15d −39b −9b −25b e e e e 1

•

45b 157b

TS

ref

84 95 76 66 42 37−71c 40 5−52b,c 52 50d 39d 7−47b,c 40b 2b 59 42 33 23 73 40 46 64 72−84c

115 349 240 240 240 240 240 97 99 99 99 101 101 244 102 102 102 102 104 104 104 106 106

a In units of kJ mol−1 from combined single-point B3LYP and PMP2/6-311++G(2d,p) energies and including B3LYP/6-31+G(d,p) zero-point vibrational energies. bThese dissociations involve stable ion−molecule complexes as intermediates. cEnergy ranges for different conformers. dN−Cα bond cleavage in the proline ring. eBDE vary depending on the formation of amide or enolimine c fragments. fβ-Ab stands for β-aminobutyric acid.

than the direct dissociation in the zwitterionic cation radical.104 This result was independent of the level of theory used in these calculations, which were carried out with perturbational (MP2) and density functional theory (B3LYP and M06-2X) methods and the 6-311++G(2d,p) basis set.104 A systematic comparison of TS energies for N−Cα bond dissociations in zwitterionic peptide cation radicals, their isomerizations to aminoketyl radicals by proton migration, and their consecutive N−Cα bond dissociations is not yet available. The calculated bond dissociation (BDE) and TS energies have been compiled259 for a series of peptide aminoketyl radicals, as shown in an updated list (Table 3). The data indicate that the unpaired electron in the amide group substantially weakens the N−Cα bond. The typical BDE for C− N bonds in neutral closed-shell molecules such as secondary amines are 330−350 kJ mol−1.174 In contrast, BDE of N−Cα bonds in aminoketyl radicals are often negative (Table 3), indicating that the intermediates have only kinetic stability due to activation energy barriers in transition states for bond cleavage. The TS energies for N−Cα bond dissociations are also very low and comparable to those for conformational changes in peptides, such as amide cis−trans isomerization260−262 or backbone rotations.101,229 The TS energies show no clear dependence on the amino acid residues flanking the dissociating N−Cα bonds and presumably reflect the changes in both the electronic structure and hydrogen bonding patterns in the peptide cation radicals along the dissociation pathway. Nevertheless, the fact that the addition of an electron and a proton to the amide bond reduces the covalent N−Cα bond strength to levels that are comparable to or lower than those of noncovalent interactions is quite remarkable and provides the

basis for the discussion of stereochemical effects linked to peptide ion conformation. 6.4.2. Effects of Peptide Ion Conformation. Stereochemical effects on ExD have been observed for diastereoisomeric peptide ions in which one of the L-amino acid residues was replaced by a D-residue and caused changes in the fragment ion relative intensities.263−265 The facile backbone dissociation in peptide cation radicals54,240 has been exploited in studies of peptide ion conformations in the gas phase that took different approaches. Several studies have relied on the known or extrapolated protein conformations in solution or solid phase and attempted to relate the dissociations observed on ExD to the condensed phase structure.117,119,235,236,266,267 Another approach that was applied to medium-size ions relied on molecular dynamics trajectory calculations of gas-phase peptide ions using force fields developed for peptides in solution.265,268 The most rigorous approach that has been limited to smaller (3−10 residues) peptide ions used electronic structure calculations of large sets of peptide ion tautomers and conformers to identify local energy minima and sort out the isomers according to their relative enthalpies or free energies calculated at the highest applicable level of theory.102−107,140,159,228 The lowest freeenergy structures were presumed to be representative of the gas-phase ions formed by electrospray and submitted to charge reduction.156 Polfer et al. studied ECD of doubly charged ions from gonadotropin releasing hormone (GnRH) variants pyro-GluHis-Trp-Ser-Tyr-Xxx-Leu-Arg-Pro-Gly, where Xxx was glycine, 265 L-tryptophan, or D-tryptophan residues. The spectra showed +• different relative intensities of z and (z + H)+ fragment ions not only for backbone cleavages next to the Xxx residue but 6709


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also for cleavages in the other parts of the peptide chain. The peptides were presumed to be protonated at the histidine and arginine residues. Molecular dynamics calculations with the AMBER force field yielded different low-energy conformers for the GnRH variants, which were a β-turn for Xxx = Gly and DTrp and an extended conformation for Xxx = L-Trp.265 Patriksson et al. studied conformers of the eikosapeptide dication Asn-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Asp-Gly-Gly-ProSer-Ser-Gly-Arg-Pro-Pro-Pro-Ser (Trp-cage) in which the Tyr3, Gln5, and Leu7 residues were either L or D enantiomers.268 One protonation site was at arginine, while the other was ambiguous, as structures with protonated Gln5 and Lys8 gave different results. The authors noted that conformers produced by Gromacs molecular dynamics trajectory calculations did not provide explanation of the backbone cleavage sites, because hydrogen bonding of the protonated residues pointed to the Pro19 amide group, which showed no exceptional propensity for N−Cα bond cleavage. To explain the data, Patriksson et al. used a statistical analysis of neutral hydrogen bonds and proposed a new model in which the electron attachment occurred in a neutral amide group that was hydrogen bonded to another neutral amide. This intermediate was presumed to isomerize by proton migration forming an intermediate consisting of a neutral aminoketyl radical and a zwitterionic enolimidate anion−cation pair.268 This so-called nonlocal mechanism214,268 was scrutinized by further experiments and electron structure calculations. An ETD study of Crizer and McLuckey used peptide dications from Lys-Ala-Ala-Ala-Lys-Ala-Ala-Ala-Lys, Lys-Gly-Gly-GlyLys-Gly-Gly-Gly-Lys, and their derivatives in which all backbone amide groups were N-methylated.269 If amide protons were involved in the backbone cleavage, N-methylation would be expected to have a major effect on the fragmentation following ET. However, the N-methylated peptide ions showed abundant N−Cα bond cleavages in the ETD spectra that were analogous to those of the nonmodified peptide ions. Thus, the effect of N-methylation was very small in contrast to what would be expected on the basis on the nonlocal model.269 A computational study of the (Ala-Ala-Val-Ala-Arg + 2H)+• cation radicals (Figure 5) addressed the energetics of proton migration in a zwitterionic intermediate that displayed a strong hydrogen bond between the aminoketyl anion radical and a proximate neutral amide group, which was further buttressed by charge solvation of the protonated arginine side chain (Scheme 11).104 The rearrangement produced an enol−imidate intermediate that was analogous to that suggested in the nonlocal Scheme 11 model. However, the enol−imidate had an extremely low-energy barrier for the reverse exothermic proton migration to the amide and was deemed dynamically unstable. The authors concluded that N−Cα bond cleavage in the enol− imidate intermediate could not compete with the nearly barrierless reverse proton migration, so the formation of such intermediates and their role in peptide backbone dissociations were put in doubt.104 An interesting observation was made by Tsybin and coworkers concerning the backbone cleavage frequency in the ECD mass spectra of peptide di- and trications derived from the transmembrane domain of the M2 protein, Ser-Ser-AspPro-Leu-ValVal-Ala-AlaSer-Ile-IleGly-Ile-Leu-His Leu-Ile-LeuTrp-Ile-Leu-AspArg-Leu (M2 TMP).267 The ECD spectrum of the triply charged ions showed periodic variations of c and z ion intensities that peaked for backbone cleavages between the Val6Val7, Ala9Ser10, Ile12Gly13,

Scheme 11

His16Leu17, Leu19Trp20, and Asp23Arg24 residues. The authors pointed out that this amphipathic peptide preferred an α-helical conformation when embedded in the lipid membrane. No conformational analysis was attempted for the gas-phase ion. The periodic variations of backbone cleavage corresponded to the variations of the hydrophilic and hydrophobic residues in the sequence with a three-residue periodicity reminiscent of that of an α-helix (3.6 residues). The periodicity was not affected by changing the electron irradiation time within 1−100 ms. The authors discussed the data within the framework of the Cornell and UW mechanisms and emphasized the role of the neutral hydrogen bond framework and amide relaxation dynamics.267 A combined ECD and ETD study of another amphipathic peptide, a Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-NH2 segment of the small protein melectin, attempted to relate the backbone cleavage frequency with the peptide ion conformation.107 The peptide prefers an α-helix conformation in solution.270 However, computational conformational analysis of the doubly charged ion that was protonated at the Lys7 and Lys8 side chains gave globular structures for the lowest-energy conformers. The conformational search involved 800 000 conformations, and the lowest-energy structures were assigned from B3LYP and cam-B3LYP energies of a subset. The lowestenergy conformer with an α-helix structure, analogous to that presumed to exist in the condensed phase, was found to be >100 kJ mol−1 less stable than the lowest-energy globular conformer of the gas-phase dication.107 The authors noted that backbone cleavages between the Leu6−Lys7 and Lys7−Lys8 residues, leading to most abundant fragment ions, occurred at amide groups that showed high unpaired electron density in the charge-reduced cation radicals, consistent with the UW model. No periodicity was observed for dissociations of doubly and triply charged ions.107 O’Connor and co-workers used time-resolved experiments and infrared heating in conjunction with ECD to study peptide ion unfolding and refolding in the gas phase.271,272 The authors pointed out that slow heating methods, such as collisional activation or heating by blackbody infrared radiation in an ion trap, occur on the time scale of hundreds of milliseconds, which is much longer than the estimated time for N−Cα bond dissociation (40 kJ mol−1 less stable than their tautomers that contained protonated c1 fragments and neutral z1• radicals. The product energies favored the c1+ + z1• channel by only a few kilojoules per mole, but the preference substantially increased upon c1+ ion isomerization from the enolimine to the amide tautomer, which had a more favorable hydrogen bonding for internal solvation of the lysine ε-NH3 group.101 6712


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A quantitative correlation of the c1+/z1+• ion intensity ratios with amino acid proton affinities was attempted by Jensen et al. for ECID of (Xxx-Lys + 2H)2+ dipeptide dications, where Xxx were the natural amino acid residues.282 The data showed a trend in that amino acid residues of increasing basicity mainly provided more abundant c1+ ions in competition with lysine z1+• ions. Figure 11 shows a plot of ln(c1+/z1+•) versus the

several key studies that provided mechanistic details as well as statistical analysis. Scheme 12

O’Connor et al. studied the formation of c+ and (c − H)+• fragment ions in ECD of the dication from hexadecapeptide Arg-Ala-Ala-Ala-Gly-Ala-Asp-Gly-Asp-Gly-Ala-Gly-Ala-Asp-AlaArg (BUSM 1) and its all-Gly-Cα-deuterated derivative (BUSM 2) containing 8 α-deuterium and 12 α-hydrogen atoms.165 An important feature of this study was that the precursor dications were mass-selected as monoisotopic (12C, 14N) species, so fragment ions differing by 1 Da could be assigned to 1H difference without interference of natural 13C and 15N isotopes. The c fragment ion series showed substantial hydrogen atom abstraction along the peptide sequence, giving (c − H)+•/c+ ion abundance ratios ∼1. A particularly large difference was found for the c10−c13 ions. H/D scrambling resulting in D loss was found to be in the 10−20% range, except for c ions from backbone cleavages C-terminal to the aspartate residues (c7, c9, and c14), which did not show loss of D despite a substantial loss of H atoms. H/D exchange in c ions was not established because the (c − D + H)+ species could not be distinguished from the (c − H) ions at a 0.0015 Da mass difference. The authors discussed the possible mechanisms and kinetic effects on the H/D exchange.165 In another study by Tsybin et al., the (c − H)+•/c+ and (z + H)+/z+• abundance ratios were found to decrease upon heating the precursor dications by collisional activation or infrared photon absorption.284 Increasing the precursor ion internal energy was thought to promote dissociation of the (c + z•)+ complexes produced by N−Cα bond cleavages284−286 and thus prevent hydrogen atom transfer.287 Likewise, resonant ejection of charge reduced ions has been shown to decrease the relative abundance of H-atom transfer products in some cases.272 This study indicated that a substantial percentage of interfragment H-atom transfers occurs on a time scale of >1 ms. A statistical analysis of a large data set of 15 000 ECD mass spectra of tryptic peptides by Savitski et al. allowed the authors to evaluate the formation of z+•, (z + H)+, and the less common (z − H)+ fragment ions depending on the nature of amino acid residues flanking the dissociating N−Cα bonds.288 The (z + H)+ species were distinguished from the (13C,15N) satellites of the z+• ions in high-resolution ECD-FTMS spectra. HR-FTMS also established that (z + 2H)+• species were not formed upon ECD. The overall occurrence of ions formed by interfragment H-atom transfer was 47%. The effect of amino acid residues on the occurrence of (z + H)+ fragments showed different rankings depending on whether the residue was at the C- or N-terminal side of the dissociating N−Cα bond. For C-terminal residues, the ranking was Gly > Asp ≈ Ser > Thr > Asn > Trp > Glu ≥ Gln > Met > Leu ≈ Ala ≥ Phe ≈ Tyr > His > Ile ≈ Val. These

Figure 11. Plot of ln(c1+/z1+•) ratios versus the amino acid gas-phase basicities in ECID spectra of dipeptide dications. Data from ref 282.

amino acid gas-phase basicities taken from the recent critical evaluation by Bouchoux.171 In addition to the general trend discussed by Jensen et al.,282 there are a few conspicuous outliers from a linear free-energy plot, ln(c1+/z1+•) = aΔGB + b, namely, for cysteine, glutamate, histidine, and arginine, that would be expected for a thermodynamically controlled proton distribution between the fragments. The reasons for the outliers were not discussed by Jensen et al. but may involve kinetic effects as well as different electronic states and internal energies of the charge-reduced ions. Extrapolation to larger peptide ions is not straightforward, because the backbone fragment basicities depend on both the nature and number of amino acid residues in the ion or neutral. For example, ETD of the (His-Ala-AlaAla-Lys + 2H)2+ dication gave c1+/z4+• = 0.11, c2+/z3+• = 4.9, and c3+/z2+• = 482, which increased with the number of amino acid residues in the c fragments, in each case favoring protonation in the more basic fragment regardless of whether it contained the histidine or lysine residue; the amino acid GB favors lysine by 5 kJ mol−1.283 Charge distribution upon ETD is more uniform for peptides containing the very basic arginine residue, which in most cases retains the charging proton. Hydrogen atom migrations between complementary c and z fragments give rise to (c − H)+• and (z + H)+ ions. These were first reported by Zubarev et al. for ECD of a 21-mer peptide ion where mass-shifted (c10 − H)+• and (z9 + H)+ fragment ions arose by bond cleavages between the Asp-Lys and Ala-Asp residues, respectively.54 The authors noted that this effect was diminished in ECD of higher charge-state peptide ions. Interestingly, the complementary (z11 + H)+ and (c12 − H)+• fragment ions were not reported. The general scheme for the formation of (z + H)+ and (c − H)+• fragment ions (Scheme 12) was formulated on the basis of experimental results of 6713


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databases provided the statistics concerning the frequency, sensitivity, and selectivity of the neutral losses.168,210,294 The common losses of neutral radicals or molecules observed in the ECD and ETD mass spectra are summarized in Table 4. These

are the residues that develop a Cα-radical center in the primary z+• ions to function as H-atom acceptors. For the N-terminal residues the ranking was Trp > Gly > Ala ≈ Ser ≥ Leu ≥ Gln ≈ Tyr > Ile ≈ Asn ≥ Met ≥ Thr ≥ Glu > Val > Asp ≈ Phe > His. In addition, amino acids in positions one or two residues remote from the cleaving N−Cα bond were found to show a much smaller effect on the variance of the (z + H)+/z+• abundance ratios. Savitski et al. also evaluated the effect of amino acid residues being present in the complementary c fragments up to five residues away from the breaking N−Cα bond to function as potential H-atom donors. The ranking pointed to the glutamine and histidine residues as the best Hatom transfer promoting residues in the c fragments. Savitski et al. provided a thermochemical interpretation of the propensity for H-atom transfer to the C-terminal residue based on C−Hα bond dissociation energies (BDE) in amino acids obtained from DFT calculations.288 A linear correlation was attempted between the (z + H)+/z+• abundance ratios and the calculated BDE, which gave a low-quality correlation coefficient of r2 = 0.53.288 The aspartate, asparagine, glutamate, and glutamine residues were also found to facilitate interfragment H-atom transfer leading to (z + H)+ ions in the ETD mass spectra of a series of nona- and decapeptides of the Ala-Ala-His-Ala-AlaXxx-X′x′x′-(Ala)1−2-Arg type, where Xxx and X′x′x′ were aspartate, asparagine, glutamate, and glutamine residues in several combinations. The interfragment hydrogen atom transfer occurred to a similar extent for cleavages between the Xxx and X′x′x′ residues, giving (z4 + H)+ ions, and at the Nterminal side of the Xxx residue, giving (z5 + H)+ ions. The effect was found to rapidly diminish for cleavages at more remote sites.283 The formation of (z + H)+ and (c − H)+• was found to be prominent in collision-induced dissociation of long-lived charge-reduced cation radicals from ETD of several other peptide ions.289−291 A thorough computational analysis of the H atom transfer in (c + z+•) complexes was carried out by Bythell, although only for glycine-containing model peptide ions [(Gly)m + nH)(n−1)+•, m = 2−5, n = 2−4].292 The main conclusion drawn by Bythell was that transfer of Hα atoms from c to z fragments was exothermic irrespective of the complex charge. The energy barriers to H-atom abstraction were found to increase with charge, which was consistent with the lower incidence of Hatom transfers in ExD of multiply charged peptide ions. Analysis of the PES also found some plausible reaction pathways for the H-atom transfer to proceed.292

Table 4. Typical Side-Chain Neutral Losses in ExD neutral loss amino acid Gly Ala Ser pSer Thr Pro Val Leu Ile Cys

Asn

Asp

Gln Glu Lys Met His Phe Tyr Arg

6.6. Loss of Side-Chain Groups

Trp

In addition to backbone cleavages, radical-induced dissociations of peptide cation radicals also result in eliminations of sidechain groups.293 These dissociations have been of keen interest, because some are indicative of particular amino acid residues, for example, distinguishing leucine and isoleucine,203 and can aid their identification as well as peptide sequence analysis (refs 204−206). Fung and Chan summarized neutral fragments being lost in the ECD mass spectra of model peptide dications with the sequence Arg-(Gly)3-Xxx-(Gly)3-Arg, where Xxx was one of the 20 natural amino acid residues.209 In addition to common combined losses of H atoms, water, and ammonia, they identified neutral losses that were specific for particular amino acid residues or their combinations with arginine. Fung and Chan also recognized that various neutral losses occurred as secondary dissociations of the z+• fragment ions and proposed mechanisms for some.209 Further analysis of spectra

a

accurate mass (Da)

elemental composition

18.0106 30.0106 96.9691 97.9769

H2O CH2O H2PO4 H3PO4

15.0240 43.0548 56.0626 29.0397 56.0626 32.9804 45.9877 90.0014 44.0136 45.02146 58.0293 43.9898 44.9977 46.0055 44.0136 58.0293 46.0055 59.0133 58.0662 71.0735 61.0117 74.0190 82.0531 83.0609

CH3 C3H7 C4H8 C2H5 C4H8 SH CH2S SCH2CONH2a CONH2 HCONH2 CH2CONH2 CO2 CO2H CO2H2 CONH2 CH2CONH2 CO2H2 CH2COOH C3H8N C4H9N C2H5S C3H6S C4H6N2 C4H7N2

106.0419 44.0374 59.0484 101.0953 116.0505 129.0579

C7H6O CH4N2 CH5N3 C4H11N3 C8H6N C9H7N

In cysteine residues capped with iodoacetamide.

were also observed in the collision-induced105,283,295,296 and infrared multiphoton dissociation spectra297 of mass-selected z+• fragment ions produced by ETD. It is of note that z+• ions belong to the category of hydrogen-deficient peptide cation radicals (section 7), and their chemistry shares some features with that of peptide cation radicals produced by methods other than ExD. Fung and Chan addressed the mechanism for loss of C3H7 and C4H8 from the z+• ions containing the leucine residues and carried out combined B3LYP and MP2 calculations on simplified des-aminoleucine N-methyl amide and des-aminoGly-Leu-N-methylamide radical models (Scheme 13).209 6714


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Scheme 13

Scheme 14

Loss of C3H7 from the des-aminoleucine N-methyl amide proceeded by β-fission that was 96 kJ mol−1 endothermic and required additional 15 kJ mol−1 in the transition state. The elimination of i-C4H8 had a comparable threshold energy (82 kJ mol−1). For loss of C3H7 from the larger radical, Fung and Chan considered two pathways. The stepwise pathway involved an exothermic (−41 kJ mol−1) migration of the leucine Cαhydrogen atom forming an Cα-radical at the leucine residue that then underwent a β-fission loss of C3H7 (Scheme 13). The TS energies for the H-atom migration (76 kJ mol−1) and β-fission (124 kJ mol−1) were moderate. Another pathway, which had been suggested previously203,298 and included a radical attack

with a pentacoordinated carbon intermediate (Scheme 13), was found to have a very high TS energy (194 kJ mol−1) and was disfavored. The competing eliminations of C3H7 and i-C4H8 were studied for larger z4+• ions from Ala-Ala-His-Ala-Leu, which were protonated at the histidine residue, and the Ala2 Cα-radical was separated from the leucine side chain by three trans-amide groups.296 The authors considered several pathways and transition states for backbone isomerizations, amide rotations, and Hα-atom transfer forming a leucine Cα-radical intermediate for the β-fission of the side chain and loss of C3H7. The polyamide backbone in the z4+• ion was found to have sufficient 6715


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Scheme 15

flexibility to allow unhindered access of the remote Ala2-Cαradical to the leucine side-chain methylene group for H-atom transfer initiating elimination of C4H8. RRKM rate constants calculated for the competing C3H7 and C4H8 eliminations were consistent with the experimental branching ratio for these channels.296 Transition states and intermediates were also studied for the elimination of CO2 from the aspartate residue in z4+• ions produced by ETD of (Ala-His-Asp-Ala-Leu + 2H)2+, which was competitive with the eliminations of C3H7 and C4H8.296 The CO2 elimination required an amide cis−trans rotation to allow access of the histidine Cα-radical to the aspartate residue. This was followed by aspartate Hα-migration forming an aspartate Cα-radical intermediate. Loss of CO2 in the final step was accompanied by carboxyl proton transfer to the aspartate amide carbonyl forming an enolamide product (Scheme 14). The calculated relative energies of the intermediates and the TS energies indicated that the Hα atom migration was the ratedetermining step with ETS = 91 kJ mol−1 relative to the starting all-trans z4+• ion. The final step, elimination of CO2, was facile (ETS = 64 kJ mol−1), and the dissociation overall was only 18 kJ mol−1 endothermic.296 Eliminations of side-chain groups from z+• ions were found to compete with cleavages of backbone CαCO bonds forming x+• and lower z+• ions. These backbone cleavages were initiated by H-atom transfers from side-chain β-positions of histidine, phenylalanine, tyrosine, tryptophan, valine, aspartate, and asparagine residues.283,297 Abundant side-chain losses were observed by O’Connor and colleagues in the ECD spectra of dications from linear peptide ions,299 as well as from macrocyclic peptides cyclo-Leu-Leu-Phe-His-Trp-Ala-Val-GlyHis, gramicidin S, and cyclosporin A.211 The side-chain losses competed with cross-ring cleavages involving multiple bond dissociations. The authors proposed several schemes for the dissociations, called the radical cascade, involving radical cyclizations and substitutions.211 Backbone dissociations of radical intermediates were later explained by Ledvina et al. for z4+• ions from ETD of Ala-Ala-His-Ala-Arg, Ala-Ala-Tyr-Ala-

Arg, and Ala-Ala-His-Trp-Arg dications.297 Radical cyclizations were found to have high transition state energies and did not compete with other reaction pathways that involved H-atom transfers from the side chains. It might be noted that radical substitutions at the amide nitrogen, also proposed by O’Connor et al.,211 were calculated to have high activation energies in small neutral amides.219 Ion−molecule reactions of fragment ions produced by ETD were studied by McLuckey and co-workers with the goal of distinguishing the z+• and c+ type ions.300 The z+• ions from several peptides were found to react with oxygen in a quadrupole ion trap to form (z + O2)+• adducts and (z − H)+ ions by HO2• elimination from the adducts. Collisional activation or thermal heating of the (z + O2)+• adducts resulted in loss of OH• radicals and other dissociations.300,301 6.7. Rearrangements in Peptide Radicals and Cation Radicals

One-electron reduction of peptide cations often results in the detection of nondissociating species. In addition to the formation of noncovalent ion−molecule complexes, electron transfer can produce nondissociating peptide radicals and cation radicals that gain stability by isomerizing to other radical structures. Elucidation of such hidden rearrangements302 requires special techniques as has been studied for a few peptide systems. A powerful technique for structure analysis relies on charge reversal by double electron transfer that converts cations to anions. This charge-reversal experiment not only allows one to detect transient neutral intermediates303 but also can be used as a selective probe, because only neutral species that have bound electronic states for the additional electron are detectable as stable anions on the microsecond time scale of the experiment. The existence of bound electronic states is readily assessed from the calculated electron affinities of putative neutral structures. The time scales for the isomerizations and lifetimes of the neutral intermediates are in the 10−7 range in the charge reversal experiments carried out with kiloelectronvolt ion beams and presently provide the fastest probe into the peptide radical kinetics. Three specific 6716


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Scheme 16

explanation for the dissociations of long-lived charge-reduced ubiquitin cation radicals.119 The arginine guanidinium radical94,95 and arginine-containing peptide radicals (Gly-Arg + H)• and (Ala-Arg + H)• were found to undergo isomerization by migration of an arginine Cα hydrogen atom onto the guanidinium radical group formed by electron transfer.97 The Cα radicals were detected as stable anions following transfer of a second electron. Analysis of the (GR + H)• potential energy surface identified a low-energy TS (ETS = 39 kJ mol−1) for the exothermic arginine Hα-atom migration(ΔH0 = −30 kJ mol−1).97 RRKM calculations were used to estimate that ca. 12% of (GR + H)• having mean internal energies of 108 kJ mol−1 isomerized on the 200 ns time scale of the charge-reversal experiments. Another hidden rearrangement was found to affect the dissociations of histidine-containing peptide radicals96,100,228 and cation radicals.102,304 Protonated peptides containing histidine residues, for example, (His-Ala-Ile + H)+, (His-AlaLeu + H)+, (Ala-Ala-His-Ala-Leu + H)+, etc., were found to form stable anions upon collisional double electron transfer from Cs atoms at 50 keV kinetic energies.100 This behavior was explained by hidden rearrangements occurring on a 120 ns time scale and involving a carboxyl proton transfer to histidine imidazolium radical group. Similar rearrangements were found to operate in histidine-containing peptide cation radicals.102,304 Scheme 16 summarizes the proposed mechanism that was based on the product identification by electron attachment,96,100,228 collision cross-section measurements of chargereduced cation radicals,156 and detailed analysis of potential energy surfaces.102,228 The histidine rearrangement is catalyzed by a proton that could be transferred from a carboxyl or protonated ammonium group in the peptide radical or cation

reactivity effects have been discovered so far concerning the proline-, arginine-, and histidine-containing peptide radicals and cation radicals. In a study of isomeric proline-containing peptide ions, collisional electron transfer from potassium and cesium atoms to (Gly-Pro + H)+ was found to produce stable (Gly-Pro + H)• radicals that were detected after conversion to stable anions.99 In contrast, charge-reversal of isomeric (Pro-Gly + H)+ ions produced only fragment anions but no molecular survivors. The existence of stable (Gly-Pro + H)• was attributed to ringopening and hydrogen-migration isomerizations forming stable radicals of substantial electron affinities allowing their detection as stable anions (Scheme 15). The (Gly-Pro + H)• radical formed by electron transfer to (Gly-Pro + H)+ was found to be unstable and spontaneously isomerize by ammonium hydrogen atom migration to the carboxyl group. The dihydroxymethyl radical can further isomerize by exothermic ring-opening and hydrogen atom migrations forming Cα radicals in the proline or glycine residues. The hydrogen atom migrations had low TS energies, which according to RRKM calculations made them kinetically feasible on the 7 × 10−7 s time scale and within the range of internal energies for the radicals formed. The ringopening and further rearrangements in proline aminoketyl radicals presumably impair N−Cα dissociations at the Cterminal side of the proline residue and explain the low frequency of such cleavages in ExD.214,281 The unusual stability of some charge-reduced peptide cation radicals formed by electron capture117,119,273 can be explained by the stabilizing radical rearrangements in proline residues. Loss of hydrogen atoms from rearranged proline radical residues, which was kinetically competitive with backbone cleavages,99 provided an 6717


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Scheme 17

or ET−no-D effects reported by McLuckey and co-workers for several histidine-containing peptide ions.110

radical. Electron transfer to the protonated histidine ring produces a C-2′-imidazolium radical intermediate that is a local energy minimum.108 Imidazolium radicals are strong bases with a topical gas-phase basicity that was calculated for the C-2′ position in 4-methyl-(2H)imidazolium radical as GB = 1018 kJ mol−1, which is comparable to the basicity of lysine and arginine residues in peptides.172 In the absence of a charged proton donor, the peptide radical follows path ii (Scheme 16), which involves a mildly exothermic migration of the carboxyl proton to form a zwitterionic intermediate consisting of an imidazoline cation radical and the carboxylate anion in a salt bridge arrangement. This step is kinetically controlled by internal rotations of the involved groups and peptide backbone refolding to allow the carboxyl group to approach the reduced histidine ring. Steric hindrance in the rotations and refolding leading to an unfavorable spatial orientation of the imidazole and carboxyl groups was found to substantially diminish the fraction of rearranged peptide radicals.100 The proton migration itself has a negligible activation energy. The zwitterion intermediate can further undergo a highly exothermic transfer of the N-3′-H proton to the carboxylate anion to finish the isomerization yielding a C-5′-imidazolium radical, which possesses a finite electron affinity (3.04 eV) to produce a stable anion upon collisional electron transfer. When a charged N-terminal ammonium group is present, it can also work as an acid to catalyze a highly exothermic protonation of the imidazolium radical along path I (Scheme 16). The presence of the rearranged histidine ring in nondissociating peptide cation radicals was supported by collisonal activation that triggered abundant loss of a C4H6N2 neutral fragment. Interestingly, collisional cross-section measurements of (AAHAL + 2H)+• cation radicals were compatible with ion structures produced by path ii rather than those by path i.156 The hidden rearrangements described in Scheme 16 provided a rational explanation of the “electron-transfer−no-dissociation”

6.8. Modified Peptide Cation Radicals

A conspicuous feature of the chemistry of hydrogen-rich cation radicals is that the dissociations are highly sensitive to structure variations and auxiliary functional groups that affect the cation radical electronic structure in the ground and excited states. A plethora of structure modifications have been investigated that involved auxiliary fixed-charge, radical, and electron trap functionalities, as well as modifications in peptide side chains and backbone groups. Particularly large effects on the chemistry of peptide cation radicals have been observed for auxiliary groups that had bound π* states for the reducing electron and could disrupt the electronic system of the modified peptide cation radical. In contrast, functional groups that are not good electron acceptors because they lack bound anionic states, such as phosphate and sulfate esters and carbohydrates, have little or no disrupting effect and are virtually nonreactive in ExD. This has been extensively utilized for locating phosphorylation,187−190 sulfation,185,195 and O- and N-glycosylation sites186,191−194 in peptides and proteins. Phosphate ester groups (pSer) were reported to affect ECD of peptide dications by reducing the frequency of backbone cleavages. This effect was attributed to noncovalent interactions of the phosphate groups preventing dissociations, but no mechanistic studies were reported.305 Other peptide modifications that did not disrupt backbone cleavages involved cysteine, methionine,196,197 and histidine oxidation,198 arginine199 and lysine methylation,200 deamidation306−308 and aspartic−isoapartic acid isomerizations,309−312 and others313 that have been successfully detected by ExD methods, as reviewed.201,314 6.8.1. Fixed-Charge Groups. Fixed-charge groups have been used to modulate the charge state of peptide ions to increase the ionization efficiency and steer fragmentation pathways.315−319 O’Connor and co-workers applied the method of Vath and Biemann315 to convert the lysine side-chain 6718


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ammonium group in Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly to a fixed-charge 2,4,6-trimethylpyridinium group (TMP tag, Scheme 17) and studied the ECD mass spectra of thus modified doubly charged ions.320 The main effect of the TMP tag was a substantial decrease of backbone cleavages and an increase of side-chain dissociations. The most abundant dissociation by far occurred in the tag and resulted in the loss of a methyl group, presumably from the intermediate trimethylpyridinium radical. The formation of minor c and z fragment ions from doubly tagged peptide ions was taken as evidence of dissociation pathways not requiring hydrogen atom transfer for backbone cleavage.320 Tagging with the bulky tris(2,4,6-trimethoxyphenyl)phosphoniummethylcarbonyl (TMPP-ac, Scheme 17) group primarily affects N-terminal peptide amino groups.316 ECD of TMPP-tagged glycopeptides showed improved sequence coverage.321 The effect of charge was investigated for singly and doubly TMPP-tagged Gly-Lys, Lys-Gly, Ala-Lys, Lys-Ala, and Gly-Arg dipeptides.98 In peptide derivatives tagged at the Nterminus, one nonprototropic charge was fixed within the TMPP group, while the other charge was provided by protonation of the basic lysine or arginine side chains. ECD of all singly tagged peptide ions showed complete dissociation in three main competitive channels. Cleavages of the backbone N−Cα bonds formed c0 and c1 fragment ions containing the TMPP charge tag, which required proton transfer from the other protonation site. In contrast, no backbone cleavages were observed in ECD of doubly tagged ions that lacked protonated residues, indicating that proton transfer was a critically important part of the N−Cα bond dissociation process. The ECD spectra of both singly and doubly tagged peptides contained abundant (TMPP + H)+ fragment ions, originating by P−CH2 bond cleavage in the TMPP tag followed by proton transfer onto the highly basic triarylphosphine. This indicated that a substantial fraction of charge-reduced ions were unstable radicals produced by electron attachment to the triarylphosphonium group. Electron structure calculations indicated that the TMPP group had a lower intrinsic recombination energy (99% H-atom transfer to the CN group forming a stable iminyl intermediate. The N−Cα bond dissociation was negligible. In contrast, peptides with the S-(2,3,4,5,6-pentafluorobenzyl) and S-benzyl 6720


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the tyrosine residue.283,297 ETD of another set of depsipeptides revealed changes in the backbone cleavage frequency in positions remote from the ester group.343 The authors attempted to qualitatively interpret the data by changes in the product stability,343 but no structure or thermochemistry studies of the peptide ions were performed. ETD of Gly-thio-Lys, Ala-thio-Lys, and Ala-thio-Arg dications in which the amide group was replaced with a thioxoamide showed a complete lack of N−Cα bond dissociations upon ETD.344 The common and dominant dissociation of these cation radicals was the loss of a hydrogen atom. The dissociation products were characterized by collision-induced dissociation (CID) multistage tandem mass spectrometry up to CID-MS5. The experimental results were in conflict with extensive ab initio and density functional theory calculations of the potential energy surface. In silico electron transfer to the precursor dications, (Gly-thio-Lys + 2H)2+, (Alathio-Lys + 2H)2+, and (Ala-thio-Arg + 2H)2+, formed zwitterionic intermediates containing thioenol anion radical and ammonium cation groups that were local energy minima on the potential energy surface of the ground electronic state. The zwitterions underwent facile isomerization by N-terminal ammonium proton migration to the thioenol anion radical group forming aminothioketyl intermediates. Combined potential energy mapping and RRKM calculations of dissociation rate constants identified N−Cα bond cleavages as the most favorable dissociation pathways, in a stark contrast to the experimental results. This discrepancy was interpreted as being due to the population upon electron transfer of low-lying excited electronic states that promoted loss of hydrogen atoms. For (Gly-thio-Lys + 2H)+•, these excited states were characterized by time-dependent density functional theory as A−C states that had large components of Rydberg-like 3s molecular orbitals at the N-terminal and lysine ammonium groups that were conducive to hydrogen atom loss.344 Peptide ions containing β-amino acid residues were found to show anomalous dissociations in ECD345 and ETD.346 Sargaeva et al. reported ECD of heavily modified peptides in which several residues were β-alanine and its 2- and 3-alkylated homologues, as well as isoaspartic acid.345 Inclusion of the βamino acid residues resulted in a dramatic decrease of backbone cleavages leading to very few N−Cβ bond dissociations. ECD of a modified substance P dication in which the Gln5 and Leu10 residues were replaced with their β-isomers showed a complete disappearance of the c4 and c9 fragments due to missing N−Cβ bond dissociations in these positions. In contrast, isoaspartic acid residues are known to undergo N−Cβ bond cleavage forming radicals that have a COOH group in the C β position.309−312 The effect of the β-amino acid residues was related to the reduced stability of β-alkyl radicals in the z fragments formed by N−Cβ bond cleavage.345 Sargaeva et al. also proposed a mechanism of backbone cleavage that involved electron attachment to N-protonated amide groups in the peptide ions.345 However, computational analysis of peptide protonation thermochemistry in model peptides indicated that N-protonated tautomers were reactive high-energy intermediates, not thermochemically stable structures.23,347 The calculated relative free energies of pentapeptide ions containing a βaminobutyric acid residue (βAb) excluded the possibility of stable ion tautomers with N-protonated amide groups being present in both solution and the gas phase.106 Computational analysis of Ala-Ala-βAb-Ala-Ala cation radicals indicated substantially lower transition-state energies for N−Cα bond

groups showed preferential N−Cα bond dissociation that outcompeted H-atom migration to the C-4 position and fluorine substituents in the phenyl ring. These computational results were used to suggest an alternative mechanism for the quenching effect on electron-based peptide backbone dissociations of EWG-containing benzyl groups.331 Peptides S-nitrosylated at cysteine residues have been reported to undergo facile loss of NO upon electron attachment that suppressed backbone dissociations.337 This effect was explained by the electron entering a σ* orbital of the S-NO group and inducing S-NO bond cleavage analogous to that in ExD of disulfides.337 Recently, electron transfer to peptide ions modified with the diazirine ring containing photoleucine (L-2-amino-4,4-azi-pentanoic acid) residue was found to trigger unusual eliminations of N2H3, N2H4, N2H5, and NH3OH neutrals that competed with backbone N−Cα bond dissociations.338 The diazirine ring was calculated to have a substantial intrinsic electron affinity (1.5 eV) and, when reduced by electron attachment, underwent multiple proton and hydrogen atom transfers forming hydroazine and the radical neutral fragments.338 6.8.3. Crown Ether and Other Noncovalent Peptide Complexes. ExD experiments have been reported that used complexes of peptide cations with neutral molecules such as water,93 methanol,158 acetonitrile, and 18-crown-6 ether (CE).159,167 The effect of CE is to specifically coordinate lysine ammonium groups339 and substantially decrease their acidity and ability to engage in proton transfer reactions. The other effect of CE−lysine complexation is that the coordinated ammonium group has an extremely low intrinsic recombination energy (

Peptide Radicals and Cation Radicals in the Gas Phase - Chemical

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