Discovery and Mechanistic Studies of Facile N-Terminal C α –C Bond Cleavages in the Dissociation of Tyrosine-Containing Peptide Radical Cations

Discovery and Mechanistic Studies of Facile N‑Terminal Cα−C BondCleavages in the Dissociation of Tyrosine-Containing Peptide RadicalCationsXiaoyan Mu,† Tao Song,† Minjie Xu,† Cheuk-Kuen Lai,† Chi-Kit Siu,*,‡ Julia Laskin,§ and Ivan K. Chu*,†

†Department of Chemistry, The University of Hong Kong, Hong Kong, China‡Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China§Fundamental Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States 99354

*S Supporting Information

ABSTRACT: Fascinating N-terminal Cα−C bond cleavages in a series of nonbasictyrosine-containing peptide radical cations have been observed under low-energycollision-induced dissociation (CID), leading to the generation of rarely observed x-type radical fragments, with significant abundances. CID experiments of the radicalcations of the alanyltyrosylglycine tripeptide and its analogues suggested that the N-terminal Cα−C bond cleavage, yielding its [x2 + H]•+ radical cation, does not involvean N-terminal α-carbon-centered radical. Theoretical examination of a prototypicalradical cation of the alanyltyrosine dipeptide, using density functional theorycalculations, suggested that direct N-terminal Cα−C bond cleavage could produce anion−molecule complex formed between the incipient a1

+ and x1• fragments.

Subsequent proton transfer from the iminium nitrogen atom in a1+ to the acyl carbon

atom in x1• results in the observable [x1 + H]•+. The barriers against this novel Cα−C

bond cleavage and the competitive N−Cα bond cleavage, forming the complementary[c1 + 2H]+/[z1 − H]•+ ion pair, are similar (ca. 16 kcal mol−1). Rice−Ramsperger−Kassel−Marcus modeling revealed that [x1 + H]•+ and [c1 + 2H]+ species are formed with comparable rates, in agreement withenergy-resolved CID experiments for [AY]•+.

■ INTRODUCTION

Gas phase fragmentations of protein and peptide (M) ions in amass spectrometerinduced by, for example, electron-capturedissociation1,2 and electron-transfer dissociation3,4form thefoundation for top-down amino acid sequencing approaches forthe rapid identification of protein components in complexbiological samples. During these processes, protonated proteinand peptide radicals ([M + nH]•(n−1)+)5,6 are generated; theirfragmentations are governed largely by the properties of theunpaired electron. Because of their importance in modernbioanalytical chemistry, considerable attention has been drawnrecently toward understanding the radical cation chemistrybehind the fragmentations of these odd-electron biomolecularions in the gas phase.7−15 Many mechanistic studies have beenperformed to deduce the gas phase fragmentations of themolecular radical cationic forms of some oligopeptides (M•+),namely, the odd-electron peptide cations with one electron lessthan the corresponding neutral molecular forms. Generatingthe M•+ species of oligopeptides in the gas phase throughclassical electron ionization or chemical ionization methods isinefficient because of the low vapor pressures of these relativelylarge and very polar biomolecules. Several methods are,however, available for the preparation of such oligopeptideradical cations in the gas phase: common techniques includecollision-induced electron transfer of transition metal/peptide

complexes;5,9,16−22 high-energy collisional excitation with atarget gas in an accelerator mass spectrometer to induceelectron transfer;23 multiphoton laser desorption/ionization ofnonvolatile, aromatic chromophore-containing small pepti-des;24 collision-induced dissociation (CID) of peptidesderivatized with conjugated free radical initiators;25 laserphotolysis of peptides derivatized with photolabile tags;26 andphotodissociation of protonated peptides.27

In general, the resulting M•+ species can undergo charge- orradical-induced dissociation pathways, which are mainlydetermined by the locations of the charge and the radical.Typically, the positive charge on M•+ is labile and exists as amobile proton along the peptide backbone. The charge-induceddissociations of M•+ ions can be minimized if they contain anarginine residue, with which the mobile proton can be localizedon the very basic guanidine group of its side chain.28 The rolesplayed by the unpaired electron in the radical-induceddissociations can be examined using M•+ species that containradicals generated initially at different sites. For example, sidechain cleavage at a tyrosine or methionine residue in M•+ canproduce a glycine residue with its α-carbon atom initially

Received: October 24, 2013Revised: March 3, 2014Published: March 28, 2014

Article

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© 2014 American Chemical Society 4273 dx.doi.org/10.1021/jp410525f | J. Phys. Chem. B 2014, 118, 4273−4281

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holding a radical, which is in π-conjugation with the adjacentamide groups via captodative stabilization.29 Homolyticcleavages of sodiated nitrate esters of serine derivatives canalso generate α-centered radicals, which undergo 1,4-hydrogenatom migrations to form various isomeric species.30 Notably,the lithiated and dilithiated forms of α-dipeptide radical cationicisomers do not interconvert.31 CID experiments have revealedthat interconversion among the three isomeric radical cations oftriglycine with the radical being generated initially on the α-carbon atoms of different residues ([G•GG]+, [GG•G]+, and[GGG•]+)obtained via side chain losses from the corre-sponding tyrosine-containing peptide radical cations ([YGG]•+,[GYG]•+, and [GGY]•+, respectively)is unfavorable,32

consistent with the high energy barriers and slow kinetics forthe interconversion among the isomers as revealed throughdensity functional theory (DFT) calculations and Rice−Ramsperger−Kassel−Marcus (RRKM) modeling.33,34 Similarresults have also been observed for the α-centered radical andπ-radical isomers of the glycylglycyltryptophan tripeptide([G•GW]+) and [GGW]•+, respectively).35 In contrast, thebarriers against the isomerization between [G•RW]+ and[GRW]•+ are relatively low as a result of the presence of thehighly basic arginine residue, resulting in facile radicalmigrations.36

The radical cations of peptides that contain aromatic aminoacid residues can also generate benzylic-like β-radicals that candelocalize into the π-systems of the aromatic side chains.37

Mechanistic studies have revealed that β-radicals in tryptophan-and tyrosine-containing peptides can induce cleavages of N−Cα

bonds along the peptide backbone to generate mainly c/z-typeproduct pairs.35,37 Similarly, the β-radical can also inducecleavages of Cα−C bonds to result in the formation of a-typeions.38,39 Statistical studies have demonstrated that cleavage ofthe Cα−C bond C-terminal to the benzylic-like β-radical isomeris preferable.26 The complementary x-type ions resulting fromthe Cα−C bond cleavages are normally not observed becausethey are unstable against further dissociation to form the z-typeions.40,41 In this paper, we report our discovery of theformation of abundant [xn−1 + H]•+ product ions throughCID of a series of tyrosine-containing peptide radicalspossessing n amino acid residues. This dissociation reactionpresumably involves cleavage of the N-terminal Cα−C bondtogether with proton transfer from the N-terminal residue tothe C-terminal fragment. We have examined the fragmentationmechanisms using low-energy CID experiments in conjunctionwith DFT calculations and RRKM modeling.

■ EXPERIMENTAL SECTIONMaterials. Fmoc-protected amino acids and the Wang resin

were purchased from Advanced ChemTech (Louisville, KY,USA). All other chemicals were supplied from Sigma−Aldrich(St. Louis, MO, USA) or Bachem (King of Prussia, PA, USA).Oligopeptides, CuII(L)(NO3)2 complexes {L = 12-crown-4, 15-crown-5, or diethylenetriamine (dien)}, and [Co(III)(salen)]Clcomplexes [salen = N,N′-ethylenebis(salicylideneaminato)]were synthesized according to procedures described in theliterature.42−44

Mass Spectrometry. All mass spectrometry experimentswere conducted using a quadrupole ion-trap mass spectrometer(Finnigan LCQ, ThermoFinnigan, San Jose, CA, USA) or atriple quadrupole linear ion trap mass spectrometer (QTRAP,AB SCIEX, Concord, ON, Canada). The molecular peptideradical cations (M•+) were generated through collision-induced

electron transfer of ternary metal−ligand−peptide com-plexes,10,16,19,45,46 with their abundances being optimizedusing different types of complex; [CoIII(salen)M]•+ for RYGand KYG; [CuII(15-crown-5)M]•2+ and [CuII(dien)M]•2+ forAYG and AY; and [CuII(12-crown-4)M]•2+ for all otherpeptides. Samples typically comprised 600 μM metal complexand 50 μM oligopeptide in a 1:1 H2O/MeOH solution. Asyringe pump (Cole Parmer, Vernon Hills, IL, USA) was usedfor direct infusion of the electrospray samples (flow rate: 30 μLh−1). CID spectra were acquired using He as the collision gas.The injection time and excitation time for CID in the ion trapwere 200 and 30 ms, respectively; the amplitude of theexcitation was optimized for each experiment. For theexperiments performed with the triple-quadrupole instrument,[CuII(L)M]•2+ was first introduced to the ion source by theelectrospray with an ion spray voltage of 3.5 kV. The M•+

species was then generated through in-source fragmentations ofthe complexes using N2 as the collision gas under a declusteringpotential of 80 eV. The resulting M•+ species was selected inthe first quadrupole (Q1) and further dissociated in thecollision cell under the MS2 mode. The collision energy was setat 5, 10, 15, 20, or 25 eV. Typically, 500 scans were summed togenerate a mass spectrum at a scan rate of 1000 Da s−1.

■ COMPUTATIONAL METHODSAll DFT calculations were performed using the Gaussian 03quantum chemical package.47 The geometries of the molecularions at the stationary points on the potential energy surface(PES) for the dissociations of [AY]•+ were optimized at theunrestricted B3LYP/6-311++G(d,p) level.48−50 All optimizedgeometries were examined through harmonic vibrationalanalyses, which give all-real frequencies for the structureslocated at local minima or one imaginary frequency for thetransition structures. The local minima associated with eachtransition structure were verified by the intrinsic reactioncoordinate method.51 Locations of the charge and radical ofeach optimized structures were identified from the atomiccharges and spin density distributions, as evaluated throughnatural population analyses.52 Relative enthalpies of theoptimized structures at 0 K (ΔH°0) were calculated fromtheir electronic energies and zero-point correction energiesobtained within the harmonic approximation. In dissociationreactions, bond cleavages led to the formation of proton-boundcomplexes formed by the incipient fragments. The energiesrequired to dissociate these proton-bound complexes into theseparated fragments were corrected with basic set superpositionerrors (ca. 0.5−1.4 kcal mol−1), which were evaluated using thecounterpoise method.The microcanonical rate constant ki(E) of each unimolecular

reaction i was calculated using the RRKM equation.54 Thevalue of ki(E) is a function of the internal energy E of thereactant, relative to the energy of the structure at the globalminimum on the potential energy surfaces (PES); it is given byeq 1

σρ

=−‡

k EW E E

h E( )

( )( )i

i i i

i i

0

(1)

where Ei = E − ΔH0i is the available vibrational energy (ΔH0i isthe ith reactant’s enthalpy of formation at 0 K), ρi(Ei) is thedensity of vibrational states of the reactant, Wi

‡(Ei − E0i) is thesum of the vibrational states of the transition state, E0i is thecorresponding critical energy for reaction, h is Planck’s

The Journal of Physical Chemistry B Article

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constant, and σ is the reaction path degeneracy, which is equalto one in all of the reaction pathways considered in this study.Activation entropies for individual reaction channels on thePES were calculated from the vibrational frequencies of thecorresponding precursor and the transition state at 450 K,along with manual inspection of the transition state frequenciesand activation entropies for the subsequently used in RRKMcalculations. Microcanonical rate constants were calculated forall possible decay channels of [AY]•+ using relative energies andvibrational frequencies obtained from DFT calculations withthe exception of k5 (in Figure S6b). The harmonic vibrationalfrequencies were used to calculate the density of states of thereactant and the sum of states of the transition structure foreach unimolecular reaction i on the PES. The correspondingmicrocanonical rate constants ki(E) were calculated using theRRKM equation,53,54 as described in detail elsewhere.55

■ RESULTS AND DISCUSSION

CID of Tyrosine-Containing Peptide Radical Cations.Figure 1a displays the CID spectrum of [RYG]•+. It featurestwo abundant product ions, a2

+ (m/z 292) and [M − 106]•+

(m/z 288), that were formed through β-radical-induced Cα−Cbond cleavage of the tyrosine residue38,39,56 and Cα−Cβ bondcleavage of the tyrosine residue with loss of neutral p-quinomethide (CH2C6H4O, 106 Da)32,57,58 and a minor signalfor the [b2 − H]•+ species (m/z 319).Figure 1b presents the CID spectrum of [KYG]•+, possessing

the relatively less-basic lysine residue at the N-terminus. In thiscase, [b2 − H]•+ (m/z 291) was the most abundant ion, withy2

+ (m/z 239) and a2+ (m/z 264) ions being the minor

products. The less-abundant a2+ ion produced through CID of

[KYG]•+, relative to the case for [RYG]•+, presumably arosebecause the less-basic lysine residue facilitates charge-inducedfragmentations.38 The CID of [NYG]•+ (Figure 1c) with an

even-less-basic N-terminal asparagine residue gave no a2+ ion

(m/z 250), with cleavage of the N−Cα bond of the tyrosineresidue resulting in the [c1 + 2H]+ ion (m/z 132) becomingdominant. Very interestingly, an uncommonly observed x-typeion was produced, namely, [x2 + H]•+ (m/z 266), whichpresumably formed through cleavage of the Cα−C bond at theasparagine residue together with a hydrogen atom transfer fromthe N-terminus to the C-terminal fragment.26,38,39 Formation ofthe present [xn−1 + H]•+ species has previously been observedin the CID of some arginine-containing radical peptide cations,but it has seemed to occur in a stochastic manner.9,26 In thisstudy, we found that systematically varying the N-terminalresidue of [XYG]•+ to an aliphatic amino acid (i.e., X = A, V, L,or I) gave increasingly more abundant [x2 + H]•+ species(Table 1). For example, the [x2 + H]•+ species (m/z 266)became the most abundant product ions from the CIDs of[AYG]•+ (Figure 1d) and [LYG]•+ (Figure S1). In contrast, theCID of protonated [AYG + H]+ species (Figure S2) resulted inmainly amide bond cleavages to produce solely b2

+ ions.Apparently, the formation of [x2 + H]•+ species was initiated

Figure 1. CID spectra of (a) [RYG]•+, (b) [KYG]•+, (c) [NYG]•+, and (d) [AYG]•+.

Table 1. Relative Abundance of [x2 + H]•+ andComplementary [c1 + 2H]+/[z2 − H]•+ Pairs Generatedthrough N-Terminal Cα−C Bond and the Tyrosine N−CαBond Cleavages, Respectively, During CID of [XYG]•+a

radicalpeptide [x2 + H]•+, %

[c1 + 2H]+, %/[z2 − H]•+, % other pathways, %

AYG 100 18/10 [b2 − H]•+, 12VYG 100 10/4 [G•YG]+, 10LYGb 100 18/0 m/z 252, 25IYG 100 18/0 [M − H2O]

•+, 12;[M − 57]+, 8

aX = A, V, L, I. bTypical CID spectrum [LYG]•+ is presented in FigureS1.



through a radical-induced process. Notably, complementary [c1+ 2H]+/[z2 − H]•+ ion pairs, resulting from the morecommonly observed N−Cα bond cleavage of tyrosine residues,were also produced in most cases (Table 1). To confirm thecompetitiveness of the formation of [x2 + H]•+, we alsoperformed CID experiments using a triple-quadrupole instru-ment, in which M•+ species were generated through in-sourcefragmentations of [CuII(15-crown-5)(M)]•2+ species; all ionsresulting from the competitive Cα−C and N−Cα bondcleavages were also clearly observed, including a1

+/[x2 + H]•+

and [c1 + 2H]+/[z2 − H]•+ ion pairs, respectively (Figure S3).We further elucidated the fundamental factors governing thisfascinating Cα−C bond cleavage by placing the tyrosine residueat different positions along peptides of various lengthsnamely, forming [AYGn−2]

•+ and [AGn−3YG]•+ (n = 4−6)

species; [xn−1 + H]•+ species were especially abundant when thetyrosine residue was in close proximity to the N-terminus, asillustrated by the CID of [AYGn−2]

•+ (Table 2).

Mechanism for the Formation of [xn−1 + H]•+ in CID ofTripeptide Radical Cations. The fragmentation reactions ofmolecular peptide radical cations are influenced by thelocations of the radical and the charge; they can occur througheither direct bond cleavages or a series of isomerizations thatmigrate the radical to different locations as intermediatesteps.9,35,36,38,59 The roles played by the radical in the formationof [xn−1 + H]•+ through N-terminal Cα−C bond cleavage havebeen elucidated using various analogues of [AYG]•+ withdifferent, but well-defined, initial radical sites and structures,including (a) [Aα

•YG]+, which features a well-defined radicalinitially located on the α-carbon atom of the alanine residue,forming a captodatively stabilized structure;29,60 (b)[AYα‑CH3G]

•+, in which the α-hydrogen atom of the tyrosineresidue was replaced by a methyl group so that the radical couldnot migrate to its α-carbon atom via abstraction of the α-hydrogen atom; and (c) [AY−CH2G]

•+, where Y−CH2 is atyrosine analogue4-hydroxyphenylglycinelacking the β-CH2 group and, hence, no benzylic-like β-radical could beformed.[Aα

•YG]+ was produced through multistage CID of[CuII(12-crown-4)(Mα‑CH3YG)]

•2+, where Mα‑CH3 is a methio-nine residue with its α-hydrogen atom replaced by a methylgroup, using methods previously discussed;32,35,36,61 briefly,collision-induced electron transfer of the metal−ligand−peptidecomplex first generated [Mα‑CH3YG]

•+, which then underwentsuccessive stages of CID to produce [Aα

•YG]+ after loss ofneutral CH2CHSCH3 from the side chain of the Mα‑CH3residue.62,63 The CID of [Aα

•YG]+ (Figure 2a) was dominatedsolely by the charge-induced dissociation to give [b2 − H]•+

(m/z 234), indicating that this N-terminal α-radical isomer wasnot involved in the formation of [x2 + H]•+ from the CID of

[AYG]•+ as shown in Figure 1d. In contrast, the CID of[AYα‑CH3G]

•+ (Figure 2b) still produced abundant [x2 + H]•+

species (m/z 280), suggesting that the α-radical at the middleresidue was not necessary for the formation of [x2 + H]•+.[AYα‑CH3G]

•+ was a π-centered radical produced directly fromthe collision-induced electron transfer of the [CuII(12-crown-4)(AYα‑CH3G)]

•2+ complex. The ion at m/z 216 was likelyformed through heterolytic Cα−Cβ bond cleavage of the Yα‑CH3residue, losing its side chain as a p-hydroxyphenylmethyl radical(107 Da). We assigned the ion at m/z 235 [an analogue of thatat m/z 221 observed in the CID spectrum of [AYG]•+ (Figure

Table 2. Observed Product Ions Generated through N-Terminal Cα−C Bond Cleavage During CID of [AYGn−2]

•+

and [AGn−3YG]•+ (n = 4−6)

peptide (M) [xn−1 + H]•+, % other pathways, %

AYGGGG [x5 + H]•+, 100 [z5 + H]•+, 28AYGGG [x4 + H]•+, 100 [z4 − H]•+, 28AYGG [x3 + H]•+, 100 [z3 − H]•+, 30AGGGYG [x5 + H]•+, 10 [c4 + 2H]+, 100; [M − 106]•+, 45AGGYG [x4 + H]•+, 10 [M − 106]•+, 100; [c3 + 2H]+, 80AGYG [x3 + H]•+, 36 [c2 + 2H]+, 100; [M − 106]•+, 30

Figure 2. CID spectra of (a) [Aα•YG]+, in which the position of the

radical is initially well-defined on the N-terminal α-carbon atom of thealanine residue; (b) [AYα‑CH3G]

•+, in which “Yα‑CH3” represents amodified tyrosine residue with the α-hydrogen atom substituted by amethyl group, preventing α-radical formation at the tyrosine residue;(c) [AY−CH2G]

•+, where “Y−CH2” is 4-hydroxyphenylglycine, ananalogue of tyrosine lacking β-hydrogen atoms.



1d)] as the [z2 − H]•+ ion produced from the benzylic-like β-radical-induced N−Cα bond cleavage of the Yα‑CH3 residue.

35,37

Formation of such a β-radical on the tyrosine residue wasalso not mandatory for the production of [x2 + H]•+ species, asrevealed in the CID spectrum of [AY−CH2G]

•+ (Figure 2c) inwhich [x2 + H]•+ (m/z 252) was again clearly observed eventhough no β-CH2 group was available in the Y−CH2 residue. Thea2

+ ion (m/z 193) observed only in the CID spectrum of[AY−CH2G]

•+ among all of these [AYG]•+ analogues most likelyoccurred because the resulting iminium moiety (−NH+CH−) produced after the Cα−C bond cleavage is stabilizedthrough π-conjugation with the phenol ring.In short, the CID spectra of [Aα

•YG]+, [AYα‑CH3G]•+, and

[AY−CH2G]•+ provided evidence for the N-terminal Cα−C

bond cleavages during CID of tyrosine-containing peptideradical cations not involving the α-carbon-centered radical ofthe N-terminal residue nor the α-carbon-centered and benzylic-like β-carbon-centered radicals of the tyrosine residue (Figure2). It is very likely that, prior to the N-terminal Cα−C bondcleavage, both the charge and radical were located on the π-system of the side chain of the tyrosine residue, with the bondcleavage being apparently more effective when the π-radical wasnear the N-terminus (Table 2). CID of [AY]•+ performed withthe triple-quadrupole instrument (Figure 3), the smallest

prototypical [AYGn−2]•+ analogue (Table 2 and Figure 1d),

also led to Cα−C bond cleavage to form the [x1 + H]•+ species(i.e., [xn−1 + H]•+ with n = 2) as well as N−Cα bond cleavage toform the commonly observed [c1 + 2H]+/[z1 − H]•+ ion pair.In fact, the formation of [x1 + H]•+ species from the

dissociations of [AY]•+ species involves a direct N-terminalCα−C bond cleavage followed by a proton transfer from the N-terminus to the C-terminal fragment, as suggested theoreticallythrough DFT calculations and RRKM modeling, which arediscussed in the following section.Energetics and Kinetics of the Dissociations of the

[AY]•+ Radical Cation. Figure 4 summarizes the PES withrespect to the N-terminal Cα−C bond cleavage (Reaction 1)and the N−Cα bond cleavage of the tyrosine residue (Reaction2) in the dissociation of [AY]•+ as obtained from DFTcalculations. [AY]•+-1 (0.0 kcal mol−1) is the lowest-energycanonical structure for [AY]•+ with both the radical and chargelocated in the π-system of the tyrosine residue, similar to thecorresponding structure obtained for the [GY]•+ analogue.37

The global minimum of the AY radical cation, with a relative

energy of −14.2 kcal mol−1, has a captodative structure inwhich the radical and proton are located on the N-terminal α-carbon and carbonyl oxygen atoms, respectively (Figure S4).This structure will not be discussed because it does notparticipate in the dissociations of the [AY]•+ radical cation, assuggested by our experimental results.

PES for the Dissociations of [AY]•+. Reaction 1: N-TerminalCα−C Bond Cleavage. The formation of [x1 + H]•+ from[AY]•+-1 first involves a direct N-terminal Cα−C bond cleavagevia a transition structure (TS1) with a relative energy of 15.3kcal mol−1, yielding COM1, which is an ion−molecule complexformed by the incipient a1

+ and x1• fragments at a shallow

minimum on the PES with a relative energy of 11.1 kcal mol−1.A similar transition state structure for the direct N-terminalCα−C bond cleavage has been reported for an analogous

Figure 3. CID spectrum of the model dipeptide [AY]•+; inset: CIDspectrum of [AY]•+ generated through collision-induced electrontransfer of [CuII(dien)(AY)]•2+ in a triple-quadrupole massspectrometer (collision energy: Elab = 5 eV).

Figure 4. Potential energy surface (PES) of [AY]•+ with respect to theformation of [x1 + H]•+/a1

+ (Reaction 1) and [c1 + 2H]+/[z1 − H]•+

(Reaction 2). Numbers are enthalpies at 0 K (all energies in kcalmol−1; bond lengths in Å). The microcanonical rate constants for eachelementary step (ki) are defined at the top.



[GY]•+ radical cation.37 The relative energy for the N-terminalCα−C bond cleavage in [GY]•+ is, however, 25.4 kcal mol−1

(data not shown), much higher than the value of 15.3 kcalmol−1 obtained for the [AY]•+-1 radical examined herein. Thisresult is in agreement with the previous experiment, in whichno [xn‑1 + H]•+ ion was observed when a glycine residue waspresent at the N-terminus.37

In COM1, a spin density of 0.93 is located on the acyl group(CO). From COM1, a proton transfer from the iminiumnitrogen atom of the a1

+ fragment to the acyl carbon atom ofthe x1

• fragment together with an electron transfer from thephenol ring to the CO group via TS2 results in COM2; inTS2, the spin density is mainly located on the acyl group (0.58)with some spin density being distributed to the phenol ring(0.19). Subsequent dissociation gives [x1 + H]•+ (m/z 209) anda neutral [a1 − H] species (43 Da) with an overallendothermicity of 21.0 kcal mol−1, which is 9.3 kcal mol−1

lower than that for the dissociation of COM1 without protontransfer, giving a1

+ (m/z 44) and the neutral radical x1• (208

Da). This energy difference seems trivial when considering thatthe delocalized π-radical on the phenol ring in [x1 + H]•+ ismore favorable than the localized σ-radical on the acyl carbonatom in x1

•. Alternative pathways for the formation of [x1 +H]•+ species that require the alanine residue to first isomerizeinto a β-carbon radical (Aβ

•) or an aminyl-radical (AN•)

structure, similar to the mechanism proposed for the Cα−Cbond cleavage of aromatic amino acid residues,38,39 are lesscompetitive. We calculated the relative energies of thetransition structures associated with these isomerizations tobe 32.7 and 21.1 kcal mol−1, respectively (see Figure S5,Supporting Information); therefore, they lie substantially aboveTS1 for the direct N-terminal Cα−C bond cleavage from[AY]•+-1.Reaction 2: N−Cα Bond Cleavage of the Tyrosine Residue.

The formation of [c1 + 2H]+ from [AY]•+-1 requires a series ofstructural rearrangements, in accordance with the analogousdissociation of [GY]•+;37 this process involves an isomerizationfrom [AY]•+-1 to [AYβ

•]+-1, a structure that contains abenzylic-like β-carbon-centered radical on the tyrosine residue,followed by sequential bond rotations to form the conformer[AYβ

•]+-2 and then [AYβ•]+-3, which can undergo subsequent

heterolytic cleavage of the N−Cα bond via the transitionstructure TS6. The energy barriers against these sequentialisomerizations are comparable, with the highest point located at15.6 kcal mol−1 (TS6), which is approximately 4 kcal mol−1

higher than that for the [GY]•+ analogue.37 COM3 is thelowest-energy ion−molecule complex of the incipient [c1 +2H]+ and [z1 − 2H]• fragments (−18.4 kcal mol−1). Directdissociation of COM3 gives [c1 + 2H]+ (m/z 89) and neutral[z1 − 2H]• (163 Da) with a relative energy of 5.3 kcal mol−1. Incontrast, dissociation with a proton transfer from the N-terminal ammonium nitrogen atom to the phenoxy oxygenatom of the tyrosine residue gives [z1 − H]•+ (m/z 164) andneutral [c1 + H] (88 Da) (i.e., an alaninamide); this process is7.4 kcal mol−1 higher in energy than the direct dissociationprocess, indicating that the proton affinity of the N-terminalmoiety is higher than that of the C-terminal moiety.35,37

Energy-Resolved CID and RRKM Modeling of theDissociation of [AY]•+. We examined the competition betweenReactions 1 and 2 of [AY]•+ through CID experimentsperformed at different collision energies using the triple-quadrupole instrument. The energy-resolved CID curves, asplotted in Figure S6a, clearly demonstrate that Reaction 2(giving [c1 + 2H]+/[z1 − H]•+) was more favorable thanReaction 1 (giving a1

+/[x1 + H]•+) at low collision energies.This order, however, reversed at center-of-mass collisionenergies higher than approximately 34 kcal mol−1, where theabundance of a1

+ rapidly increased with respect to the collisionenergy.We also performed RRKM modeling based on our proposed

mechanisms for Reaction 1 (direct N-terminal Cα−C bondcleavage) to form COM1 and COM2 leading to a1

+ and [x1 +H]•+, respectively, and Reaction 2 (N−Cα bond cleavage via aseries of structural rearrangements) to form COM3 leading to[c1 + 2H]+. We evaluated the microcanonical rate constants (ki)for elementary reactions i, as defined in Figure 4, using therelative enthalpies and the harmonic vibrational frequencies ofthe relevant structures obtained from the DFT calculations.Figure 5a,b display the calculated microcanonical rateconstants; Figure S6b presents the corresponding breakdowncurves. The breakdown curves representing the relativeabundances of the precursor ion and major fragments as a

Figure 5. RRKM modeling for (a) the two competitive fragmentation pathways of [AY]•+ to form the incipient ion−molecule complexes COM1(red circles) and COM3 (black squares); (b) the isomerization from COM1 to COM2 (blue triangles) and the dissociation of the ion−moleculecomplexes to form a1

+ (red circles), [x1 + H]•+ (purple inverted triangles), and [c1 + 2H]+ (black squares).



function of the internal energy of [AY]•+-1 were calculated bynumerically solving the rate equations of all reactions shown inScheme S1 for a reaction time of 100 ms.Figure 5a displays the overall rate constants37 for the

dissociations of [AY]•+-1 to COM1 (kAY‑1→COM1) and COM3(kAY‑1→COM3), where

=

=+ + +

− →

− →

− − − − − −

k k

kk k k k

k k k k k k k k k k k k( )

AY 1 COM1 1

AY 1 COM32 3 4 5

2 3 4 2 3 5 2 4 5 3 4 5

(detailed mathematical derivations of these two rate constantsare available in the Supporting Information). RRKM modelingsuggests that the rates of formation of COM1 and COM3 arecomparable, with the latter being only slightly (less than threetimes) faster at an internal energy of less than 100 kcal mol−1,as revealed in Figure 5a. Subsequent dissociation of COM3 at arate (kCOM3→c1+2H) similar to that of its formation (kAY‑1→COM3)results in [c1 + 2H]+ (Figure 5b). After COM1 is formed, it candirectly produce a1

+. A competing pathway is proton transfer(kCOM1→COM2) resulting in COM2, which dissociates at a muchfaster rate to form [x1 + H]•+ (kCOM2→x1+H) (Figure 5b). Theseresults indicate that, at low internal energies, Reaction 2,resulting in formation of [c1 + 2H]+, is more favorable thanReaction 1, leading to a1

+ and [x1 + H]•+. The calculatedbreakdown curves in Figure S6b further support this assertion.According to these calculations, the [c1 + 2H]+ ion is thedominant fragment at internal energies of less than 30 kcalmol−1. At higher internal excitation energies (40−80 kcalmol−1), the formation of this fragment occurs in competitionwith the formation of the [x1 + H]•+ ion, while the a1

+ ion isobserved as a dominant fragment at internal energies of greaterthan 100 kcal mol−1. The increase in the relative abundance ofthe a1

+ ion at high internal excitation is a direct consequence ofthe rapid increase in the rate of direct dissociation of COM1 toa1

+ (kCOM1→a1) upon increasing the internal energy of the ion.Notably, the abundance of the [z1 − H]•+ fragment is very lowat all internal energies.In summary, RRKM calculations predict that [c1 + 2H]+ is

the major low-energy fragment of [AY]•+-1. At intermediateinternal excitations, this fragment is formed in competition with[x1 + H]•+, while the a1

+ ion is an abundant fragment at highinternal energies. Direct comparison of these results with theexperimental data requires accurate characterization of theinternal energy distribution of the ions in the CID experiments;such a study is outside the scope of this paper. Therefore, welimit our discussion to a qualitative comparison of theexperimental and simulated breakdown curves. Collisionenergy-resolved CID data (Figure S6a) reveal that only [c1 +2H]+ and [x1 + H]•+ formed at low collision energies, with [c1+ 2H]+ being the dominant fragment. This finding agreesqualitatively with the fragment distribution at an internal energyof approximately 60 kcal mol−1 (Figure S6b). The decrease inthe relative abundance of these primary fragments accompaniedby the rapid increase in the relative abundance of the a1

+ ionupon increasing the collision energy and the low abundance ofthe [z1 − H]•+ fragment observed experimentally are in goodqualitative agreement with the results from the RRKMcalculations.In summary, DFT examinations suggested that the formation

of the competitive [x1 + H]•+ species involves direct N-terminal

Cα−C bond cleavage to yield the ion−molecule complexCOM1 formed between the incipient a1

+ and x1• fragments,

followed by a proton transfer from the iminium nitrogen atomof a1

+ to the acyl carbon atom of x1• to form COM2, the

precursor ion−molecule complex of [a1 − H] and [x1 + H]•+.This proposed mechanism is supported by RRKM modelingand the energy-resolved CID data of [AY]•+.

■ CONCLUSIONWe have discovered a novel N-terminal Cα−C bond cleavage,leading to the formation of [xn−1 + H]•+ peptide fragments, inthe low-energy CID of the radical cations of a series of nonbasictyrosine-containing peptides possessing n amino acid residues,including [XYG]•+ (X = A, V, L, I, N), [AYGn−2]

•+ (n = 2−6),and [AGn−3YG]

•+ (n = 4−6). We examined the mechanism offormation of the [xn−1 + H]•+ species using different analoguesof [AYG]•+. The [x2 + H]•+ species was absent in the CIDspectrum of [Aα

•YG]+, probably indicating that the N-terminalα-carbon-centered radical was not involved in its formation.The CID of [AYα‑CH3G]

•+ and [AY−CH2G]•+ still produced [x2

+ H]•+ species, suggesting that the α-carbon-centered andbenzylic-like β-carbon-centered radicals on the tyrosine residueare not necessary for the Cα−C bond cleavage. Energy-resolvedCID experiments, DFT calculations, and RRKM modeling ofthe dissociations of [AY]•+ revealed that the formation of [x1 +H]•+ involves direct N-terminal Cα−C bond cleavage from theπ-centered radical isomer, and that its rate is comparable withthat of the competitive N−Cα bond cleavage, forming the [c1 +2H]+/[z1 − H]•+ ion pair.

■ ASSOCIATED CONTENT*S Supporting InformationCID spectra of [LYG]•+ and [AYG + H]+; CID spectrum of[AYG]•+ obtained from the triple-quadrupole mass spectrom-eter; lowest-energy structure for the captodative [A•Y]+;transition structures for the alternative pathways for theformation of [x1 + H]•+ from [AY]•+; energy-resolved CIDof [AY]•+ performed with a triple-quadrupole mass spectrom-eter; detailed mathematical derivations of the equations for therate constants; rate equations for the dissociation of [AY]•+

expressed in terms of the RRKM rate constants. Cartesiancoordinates for all structures in Figure 4. This material isavailable free of charge via the Internet at http://pubs.acs.org/.

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: [email protected]. Telephone: (852)28592152.*E-mail: [email protected]. Telephone: (852)34422272.Author ContributionsXiaoyan Mu, Tao Song, and Minjie Xu contributed equally tothis work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the Hong Kong Research Grants Council (RGC),Hong Kong Special Administrative Region (HKSAR), China,for financial support (HKU7016/13P and HKU7016/11P).X.Y.M. and C.K.L. thank the Hong Kong RGC for supportingtheir studentships. C.K.S. thanks RGC (CityU 103110) andCity University of Hong Kong for financial support (project no.7004021). J.L. acknowledges support from the U.S. Depart-



http://pubs.acs.org/

mailto:[email protected]

mailto:[email protected]

ment of Energy, Office of Basic Energy Sciences, Division ofChemical Sciences, Geosciences & Biosciences. Pacific North-west National Laboratory (PNNL) is a multiprogram nationallaboratory operated for DOE by Battelle. We also thank Dr.Qiang Hao (City University of Hong Kong) for helpfuldiscussions.

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Discovery and Mechanistic Studies of Facile N-Terminal C α –C Bond Cleavages in the Dissociation of Tyrosine-Containing Peptide Radical Cations