Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Fragmentation of Protonated Oligoalanines:Amide Bond Cleavage and Beyond

Alex G. Harrison and Alex B. YoungDepartment of Chemistry, University of Toronto, Toronto, Ontario, Canada

The fragmentation reactions of the singly-protonated oligoalanines trialanine to hexaalaninehave been studied using energy-resolved mass spectrometry in MS2 and MS3 experiments. Theprimary fragmentation reactions are rationalized in terms of the bx-yz pathway of amide bondcleavage which results in formation of a proton-bound complex of an oxazolone and apeptide/amino acid; on decomposition of this complex the species of higher proton affinitypreferentially retains the proton. For protonated pentaalanine and protonated hexaalanine themajor primary fragmentation reaction involves cleavage of the C-terminal amide bond to formthe appropriate b ion. The lower mass b ions originate largely, if not completely, by furtherfragmentation of the initially formed b ion. MS3 energy-resolved experiments clearly show thefragmentation sequence bn 3 bn�1 3 bn�2. A more minor pathway for the alanines involvesthe sequence bn3 an3 bn�13 bn�2. The a5 ion formed from hexaalanine loses, in part, NH3to begin the sequence of fragmentation reactions a53 a5*3 a4*3 a3* where an* � an � NH3. Thea3* ion also is formed from the b3 ion by the sequence b3 3 a3 3 a3* with the final step beingsufficiently facile that the a3 ion is not observed with significant intensity in CID mass spectra.A cyclic structure is proposed for the a3* ion. (J Am Soc Mass Spectrom 2004, 15, 1810–1819)© 2004 American Society for Mass Spectrometry

With the advent of soft ionization techniquessuch as electrospray ionization (ESI) [1–3]and matrix-assisted laser desorption-ioniza-

tion (MALDI) [4, 5] which efficiently ionize by proto-nation a wide variety of peptides and proteins, tandemmass spectrometry [6, 7] has become a method ofsignificant importance for the sequencing of peptidesthrough collision-induced dissociation (CID) studies.As a result, the main types of fragmentation reactionsoccurring are well-established [8–11], at least in aphenomenological sense and are outlined in Scheme 1.A major mode of fragmentation in many cases involvescleavage of an amide bond in the protonated peptide.When the charge is retained by the C-terminus frag-ment migration of a labile hydrogen from the N-terminus neutral fragment occurs to form a protonatedamino acid (y1) or peptide (yn) [12, 13]. When the chargeremains on the N-terminus fragment, a neutral aminoacid or peptide is eliminated and bn ions are formed.Although these bn ions were initially considered to beacylium ions [8, 10, 11], extensive experimental andtheoretical studies [14–19] have shown that, in manycases, the bn ions have protonated oxazolone structureas shown in Scheme 2 formed by cyclization involving

Published online November 2, 2004Address reprint requests to Dr. A. G. Harrison, Department of Chemistry,
University of Toronto, 80 George Street, Toronto, Ontario M5S 3H6,Canada. E-mail: [email protected]
© 2004 American Society for Mass Spectrometry. Published by Elsevie1044-0305/04/$30.00doi:10.1016/j.jasms.2004.08.015

the carbonyl function next-nearest to the amide bondbeing ruptured. In several cases where there are morereactive side-chain groups in the peptide, alternativecyclic structures are formed by interaction with side-chain functionality as the amide bond cleavage occurs[20, 21].

Although the fragmentation reactions of b2 ions havebeen studied in some detail [14, 18, 22, 23], the fragmen-tation reactions of larger b ions have seen less study [15]and the fragmentation of an ions have seen even lessstudy. A related question is whether, for larger proton-ated peptides, the lower mass b ions are formed directlyby fragmentation of the protonated peptide or aresecondary fragmentation products. In the present workwe have undertaken a detailed study of protonatedoligoalanines with two goals in mind, first, to determineas far as possible the relative importance of primarycleavage of the different amide bonds in the peptideand, second, to elucidate further the fragmentationpathways of the various b ions and a ions which areobserved in the CID mass spectra. The fragmentationreactions of various protonated oligoalanines have seenextensive study [15, 24–29] although little attention hasbeen paid to the question of the relative propensity forcleavage of the different amide bonds, and the second-ary fragmentation reactions have seen only limitedattention [15]. The oligoalanines are of particular inter-est in the present context because Paizs and Suhai [29]have recently reported ab initio calculations of theproton affinities of the oxazolones and amino acids/
peptides involved in the fragmentation of protonated
r Inc. Received May 17, 2004Revised August 26, 2004

Accepted August 26, 2004

1811J Am Soc Mass Spectrom 2004, 15, 1810–1819 FRAGMENTATION OF PROTONATED OLIGOALANINES

pentaalanine which allows one to relate the fragmenta-tion reactions observed for the various oligoalanines tothe relative thermochemistry involved.

Experimental

Initial CID studies were carried out using an electro-spray/quadrupole mass spectrometer (VG Platform,Micromass, Manchester, UK) with CID in the interfacebetween the atmospheric pressure source and the quad-rupole mass analyzer, so-called cone-voltage CID. It hasbeen clearly established [30–34] that, by varying thefield in this interface region, energy-resolved massspectra [35] similar to those obtained by variable low-energy CID in quadrupole cells can be obtained. Theresults of these cone-voltage CID experiments are pre-sented in the following as breakdown graphs express-ing the percentage of total ion signal as a function of thecone voltage. MS2 and MS3 experiments were alsocarried out using an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer (QStar, MDS Sciex,Concord, Canada). In the MS3 experiments, CID in theinterface region produced fragment ions; those of inter-est being mass-selected by the first quadrupole massanalyzer (Q) to undergo collisional activation in thequadrupole collision cell (q) with the ionic fragmenta-tion products being analyzed by the time-of-flight ana-lyzer [36, 37]. By varying the collision energy in thequadrupole cell, breakdown graphs for selected frag-ment ions were obtained under multiple collision con-ditions.

Ionization was by electrospray on both instruments.For the single quadrupole the peptide was dissolved in1:1 CH3CN/1% formic acid and introduced into thesource at a flow rate of 30 �L min�1 with nitrogen asboth nebulizing and drying gas. For the QTOF instru-ment the peptide was dissolved in 1:1 CH3OH/0.1%formic acid and introduced into the source at a flowrate of 80 �L min�1. Nitrogen was used as nebuliz-ing gas, drying gas and collision gas in the quadrupolecell.

All peptide samples were obtained from BACHEMBiosciences (King of Prussia, PA) and were used asreceived.

Results and Discussion

The detailed mechanism of amide bond cleavage in

Scheme 1

protonated peptides has not been definitively estab-

lished although there is increasing evidence [29,38 – 41] that fragmentation occurs from the amideN-protonated species to form, at low internal ener-gies, a proton-bound complex of an oxazolone and atruncated peptide or amino acid as illustrated inScheme 2, the so-called bx-yz pathway [41]. Thefragmentation of such a proton-bound complexshould be determined, largely by the relative protonaffinities of the oxazolone and the peptide/aminoacid, with that having the greater proton affinitybeing preferentially formed as the ionic species. Theresults of the present study will be interpreted interms of the pathway of Scheme 2 and, indeed,provide support for the pathway.

Trialanine

The CID mass spectra of protonated trialanine obtainedat three collision energies in the quadrupole collisioncell are presented in Table 1. At the lowest collisionenergy only the b2 fragment ion is observed indicatingpreferred fragmentation of the C-terminal amide bond.That the b2 ion should be formed rather than the y1 ionis in agreement with the relative proton affinities, thatfor the neutral b2 oxazolone being 222.3 kcal mol�1

compared with 214.6 kcal mol�1 for alanine [29]. With

Scheme 2

increasing collision energy fragmentation of the b2 ion

by cone-voltage CID. Ion signal for MH� not shown.

1812 HARRISON AND YOUNG J Am Soc Mass Spectrom 2004, 15, 1810–1819

to the a2 ion is observed as well as formation of the y2

ion arising from cleavage of the N-terminal amidebond. Laskin and co-workers [26–28] have examinedthe fragmentation of protonated trialanine by surface-induced dissociation (SID) and by multiple-collisionactivation (SORI) using Fourier transform/ion cyclo-tron resonance (FT/ICR) mass spectrometry. Ourpresent results are in best agreement with their SIDspectrum, particularly with respect to the intensity ofthe y2 ion. As they have noted [26], multiple-collisionactivation in FT/ICR discriminated against processes ofhigher critical reaction energy, in this case formation ofthe y2 ion.

Tetraalanine

The CID mass spectra of protonated tetraalanine atthree collision energies are given in Table 2 while thebreakdown graph obtained by cone-voltage CID ispresented in Figure 1. The breakdown graph for the b3

ion obtained on the QTOF instrument is presented inFigure 2. Clearly, the b3 and y2 ions are the majorprimary fragmentation products. An earlier metastableion study [15] of the fragmentation of protonated tet-raalanine produced by fast atom bombardment (FAB)showed y2(100), b3(52), and �H2O(14). It is clear thatthe majority, if not all, of the b2 ion arises by furtherfragmentation of the b3 ion; this is in agreement withthe conclusions of Laskin et al. [27]. (We cannot pre-clude the possibility that some of the b2 ion arises bydirect fragmentation of the central amide bond at high

Table 1. CID mass spectra of protonated trialanine

m/z Ion

Collision energy

6 eV 10 eV 14 eV

232 MH� 100 55.9 10.1214 �H2O 2.1 1.1161 y2 8.4 21.8143 b2 23.6 100 100115 a2 11.1 37.490 y1 2.1

Table 2. CID mass spectra of protonated tetraalanine*

m/z Ion

Collision energy

6 eV 15 eV 20 eV

303 MH� 100 22.5 2.1285 �H2O 1.2 3.9232 y3 5.6 6.6214 b3 6.4 43.0 25.2169 a3* 11.8 19.3161 y2 11.4 100 100143 b2 1.5 38.4 85.0115 a2 8.3 29.744 a1 3.5

*a3* � a3 � NH3

Figure 1. Breakdown graph for protonated tetraalanine obtained

Figure 2. Breakdown graph for the b ion derived from tet-
3
raalanine.


collision energies even though this is the thermochemi-cally disfavored product; see below.) Figures 1 and 2clearly indicate the reaction sequence b33 b23 a2; thissequence is also evident from the breakdown graphobtained earlier [15] for protonated tetraalanine. Cleav-age of the C-terminal amide bond leads to formation ofthe b3 ion rather than the y1 ion since the proton affinityof the corresponding oxazolone is 226.0 kcal mol�1

compared with 214.6 kcal mol�1 for alanine [29]. Simi-larly, cleavage of the central amide bond leads primar-ily to formation of the y2 ion since PA(AA) � 225.4 kcalmol�1 compared with 222.3 kcal mol�1 for the neutraloxazolone corresponding to the b2 ion. An earlier meta-stable ion study of the fragmentation of the b3 ion

Figure 3. (a) CID mass spectrum of protonateenergy. ax* � ax � NH3. (b) CID mass spectrumCID at 40 V cone voltage.

produced from pentaalanine showed formation of

a3(81), a3*(100) and b2(71), where a3* � a3-NH3; as shownin Figure 2, the a3 ion is barely observed in the CIDmass spectrum of the b3 ion while the a3* ion is observedin relatively low abundance. The formation of an* ionswill be discussed in detail below.

Pentaalanine

Figure 3 compares the CID mass spectrum for proton-ated pentaalanine obtained with the QTOF instrumentwith that obtained by cone-voltage CID on the singlequadrupole instrument. The spectra are quite similarand are in agreement with the spectra reported byLaskin and Futrell [27], particularly those obtained by

taalanine obtained on QTOF at 20 eV collisiontonated pentaalanine obtained by cone-voltage

d penof pro

surface-induced dissociation. The breakdown graph for


MH� obtained by cone-voltage CID is presented inFigure 4. Clearly, formation of b4 is the most prominentfragmentation reaction with minor formation of y3, b3,b5 (-H2O), and y2. We believe that the apparent higheronset for the y2 product in Figure 4 is not real butreflects the detection limit for low-intensity ions. Meta-stable ion fragmentation of protonated pentaalanineshowed b5(34), b4(100), b3(22), y3(28), and y2(8) asfragmentation products [15]. If there was an equalpropensity for cleavage of the various amide bonds, onewould have expected the yields of b3, y3, and y2 to begreater in both the metastable ion and lowest energyCID mass spectra. The dominance of the b4 ion in bothindicates that the C-terminal amide bond is preferen-tially cleaved. The calculations of Paizs and Suhai [29]have shown that the energy requirement for transfer ofthe proton from the most-favored amino position to the

Figure 4. Breakdown graph for protonated pentaalanine ob-tained by cone-voltage CID. MH� ion signal not shown.

C-terminal amide nitrogen is lower than for transfer to

other amide nitrogens and that the energy requirementfor cleavage of the C-terminal amide bond is lower thanfor cleavage of other amide bonds.

The primary fragmentation reactions of protonatedpentaalanine can be rationalized in terms of the relativeproton affinities of the respective products expected bythe bx-yz mechanism outlined in Scheme 2 and, indeed,provides support for the fragmentation mechanism.The proton affinities of the relevant oxazolones andamino acid/peptides, calculated by Paizs and Suhai[29], are presented in Table 3. Clearly, cleavage of theC-terminal amide bond will lead to formation of the b4

ion since the b4 oxazolone has a much higher protonaffinity than that of alanine (y1); this is what is observedin both metastable ion and CID mass spectra. On theother hand, cleavage of the next amide bond shouldlead to formation of both b3 and y2 ions since the protonaffinities of the corresponding neutral are very similar;thus, both b3 and y2 are observed in metastable ionfragmentation. Cleavage of the b2™y3 bond should re-sult in formation of the y3 ion rather than the b2 ionsince the proton affinity of trialanine is considerablygreater than that of the b2 oxazolone. The dominance ofthe b3 ion (with respect to the y2 ion) and the abundantion signal for the b2 ion (Figure 3) arise from thedominant primary cleavage of the C-terminal amidebond to form the b4 ion which fragments, in part, by thesequence b4 3 b3 3 b2. This sequence is shown by thebreakdown graph for the b4 ion presented in Figure 5.Formation of the a4 ion (CO loss) and the b3 ion are themajor low-energy fragmentation routes of the b4 ion; ametastable ion study [15] of the b4 ion showed a4(100),b3(38), and b2(7). It is evident that under CID conditionsthe b3 ion is more readily formed. In the earlier study itwas proposed that the neutral accompanying the bn 3bn�1 fragmentation was a cyclic aziridinone, however,subsequent ab initio calculations [42, 43] have shownthat this is a very energy-demanding route whichcannot compete with the stepwise process bn 3 an 3bn�1. Thus, the detailed pathway for the direct bn 3bn�1 reaction is not known; a possibility is outlined inScheme 3. That the reaction sequence b43 a43 b3 does

Table 3. Thermochemistry for fragmentation of protonatedpentaalanine

N-Terminus C-Terminus

Ion PA(Neut)a Ion PA(Neut)a

b4 232.6 y1 214.6b3 226.0 y2 225.4b2 222.3 y3 233.1

akcal mol�1

occur is shown by the formation of the a4 ion from b4


(Figure 5) and by the breakdown graph for the a4 ion(Figure 6) which clearly shows the sequence a43 b33b2 3 a2. The likely pathway for the an 3 bn�1 reactionis shown in Scheme 4. The other fragmentation path-way for the a4 ion is loss of NH3 to form the a4* ion. Aprominent ion in the breakdown graph of Figure 6 is thea3* ion which appears to be formed both by loss of analanine residue from the a4* ion and by loss of CO �NH3 from the b3 ion (see above). The a3* fragmentsfurther by loss of CO to give m/z 141 with further minorloss of CO to give m/z 113. Possible routes to andstructures of the an* ions are discussed later.

Hexaalanine

The CID mass spectrum of protonated hexaalanine,obtained on the QTOF instrument, is shown in Figure 7.The bn ion series dominates the mass spectrum withmore minor yn ion series and an ion series. Of particularnote is the absence of the a3 ion and the significantintensities for the an* series of ions. The breakdowngraph for the MH� ion, obtained by cone-voltage CID,is shown in Figure 8. Clearly, the b5 ion is the most

Figure 5. Breakdown graph for the b4 ion derived from penta-alanine.

Scheme 3

abundant fragment ion at low collision energies withmore minor yields of b4, y4, and y3; the y2, b3, and b2

ions are secondary products. Thermochemical data forfragmentation of protonated hexaalanine are presentedin Table 4. (The proton affinities of the b5 oxazolone andthe y4 peptide have not been calculated and are as-sumed to be equal to or greater than those for the

Figure 6. Breakdown graph for the a4 ion derived from penta-alanine.

Scheme 4

tained on QTOF at 20 eV collision energy.

by cone-voltage CID.


homologous b4 and y3 species.) The thermochemicaldata indicate that formation of b5 should be favoredover formation of y1, formation of b4 favored overformation of y2 but formation of y3 and y4 should befavored over formation of the complimentary b3 and b2

ions; this is what is observed experimentally (Figure 8),thus providing support for the bx™yz pathway outlinedin Scheme 2. If there were an equal propensity forcleavage of the various amide bonds, one would expectto see more pronounced ion signals for b4, y4, and y3 atthe lowest cone voltages; the relative dominance of theb5 ion indicates that there is a preference for cleavage ofthe C-terminal amide bond as was observed for proton-ated pentaalanine.

The preferential cleavage of the C-terminal amidebond to form b5 and the fragmentation sequence b5 3b43 b33 b2, which is clearly shown by the breakdowngraph for the b5 ion (Figure 9), accounts for the domi-nance of the b ion series in the CID mass spectrum ofFigure 7. A second, more minor, fragmentation routefor the b5 ion forms the a5 ion (CO loss) which initiatesthe fragmentation a5 3 a4 and the sequence a5 3 a5* 3a4* 3 a3*, with the latter becoming a prominent productat higher collision energies. This fragmentation se-quence is shown clearly by the breakdown graph for thea5 ion (Figure 10) where the an* series of ions dominatethe breakdown graph along with more minor formationof the b4 ion as a primary fragmentation product. Theorigin of and possible structures of the an* ions arediscussed below.

Formation of an* Ions

The CID mass spectra of protonated tetraalanine tohexaalanine show significant ion signals for an* (an �NH3) ions. The only free amino group available to belost as NH3 is the N-terminal amino function. Loss ofthis group in the absence of any rearrangement wouldlead to an electron-deficient carbocation [44] which isdestabilized by the �-carboxy group; such species areknown [45–48] to have limited stability in the gas-phase

Table 4. Thermochemistry for fragmentation of protonatedhexaalanine

N-Terminus C-Terminus

Ion PA(Neut)a Ion PA(Neut)a

b5 �232.6 y1 214.6b4 232.6 y2 225.4b3 226.0 y3 233.1b2 222.3 y4 �233.1

akcal mol�1

Figure 7. CID mass spectrum of protonated hexaalanine ob-

Figure 8. Breakdown graph for protonated hexaalanine obtained
and we suggest that cyclization has occurred upon loss


of NH3. The most straightforward case is the formationof the a3* ion in the mass spectrum of protonatedtetraalanine. Earlier metastable ion studies [15] indicatethe reaction sequence MH�3 b33 a33 a3*; under CIDconditions, the last step appears to be particularly facilesince the a3 ion abundance is very low in the CID massspectrum. In Scheme 5 we propose a pathway for the a3

3 a3* reaction leading to a cyclic structure which mightbe expected to lose CO on further fragmentation, asobserved experimentally.

The breakdown graph for the a5 ion derived fromhexaalanine (Figure 10) clearly shows the reaction se-quence a53 a5*3 a4*3 a3*, i.e., the initial loss of NH3 isfollowed by sequential loss of two alanine residuesresulting in formation of the stable a3* ion. It is not clearwhether the a5* and a4* species are cyclic or remain in theacyclic form; certainly, if they are larger cyclic ions theymust open to accommodate the loss of the alanine

Figure 9. Breakdown graph for the b5 ion derived fromhexaalanine.

residues.

Conclusions

The primary fragmentation reactions of the protonatedoligoalanines involve cleavage of an amide bond toproduce bx or yz ions. The present work has shown thatthe primary fragmentation reactions can be adequatelyrationalized in terms of the bx™yz pathway outlined inScheme 2, with the relative abundances of the bx and yz

ions being determined by the relative proton affinitiesof the corresponding neutral oxazolones and peptides/amino acid. The results thus provide strong support forthe fragmentation mechanism outlined in Scheme 2. Forprotonated pentaalanine and protonated hexaalaninethere is a distinct preference for primary fragmentationby cleavage of the C-terminal amide bond to form therespective b ion. The majority, if not all, of the lowermass b ions originate by sequential fragmentation of thebn ion formed by cleavage of the C-terminal amidebond. The major fragmentation pathway for the oli-

Figure 10. Breakdown graph for the b5 ion derived fromhexaalanine.

goalanines is the sequence bn 3 bn�1 3 bn-2 with a


minor contribution from the sequence bn 3 an 3 bn�1.The fact that most of the lower mass b ions are second-ary products has significant implications for the condi-tions used to record CID mass spectra, if one wishes toobserve the complete series of b ions, which are often ofmajor importance in peptide sequencing. The behaviorobserved for the oligoalanines will undoubtedly changewhen amino acids with different functionalities arepresent in the peptide, since they will affect not only thefavored site of amide nitrogen protonation but also therelative proton affinities of the oxazolones and peptideswhich will be reflected in the products formed by amidebond cleavage. In addition, they likely will have adistinct influence on the relative importance of the bn3bn�1 3 bn�2 and bn 3 an 3 bn�1 pathways. Theseaspects are under study in our laboratory.

AcknowledgmentsThis work was supported by the Natural Sciences and Engineer-ing Council (Canada) through a research grant to AGH and anequipment grant to the Department of Chemistry which madepossible the purchase of the QStar. AGH is indebted to Dr. B. Paizsfor helpful discussions and the communication of results prior to

Scheme 5

publication.

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Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond