Fragmentation reactions of some peptide b3 ions: an energy-resolved study

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2009; 23: 1298–1302

) DOI: 10.1002/rcm.4003
Published online in Wiley InterScience (www.interscience.wiley.com
Fragmentation reactions of some peptide b3 ions:

an energy-resolved study

Alex G. Harrison*Department of Chemistry, University of Toronto, Toronto, Canada

Received 15 January 2009; Revised 25 February 2009; Accepted 26 February 2009

*CorrespoUniversit3H6, CanE-mail: aContract/Research

The fragmentation reactions of b3 ions of nominal structure AAAoxa, YAAoxa, AYAoxa and AAYoxa

have been studied as a function of collision energy, allowing the construction of breakdown graphs

expressing in a qualitative way the energy dependence of the fragmentation reactions. The primary

fragmentation reactions of the AAAoxa b3 ion involve formation of the a3� (a3 – NH3) ion and the b2

ion, with the latter becoming the dominant product at higher internal energies. For both YAAoxa and

AYAoxa b3 ions the pathway to a3� is relatively minor with formation of b2 the dominant primary

fragmentation reaction. For the AAYoxa b3 ion, in addition to a3�, abundant formation of the tyrosine

(Y) iminium ion is observed with only minor formation of the b2 ion. The results support and expand

upon the detailed mechanism of fragmentation of b3 ions proposed by Cooper et al. (J. Am. Soc.Mass

Spectrom. 2006; 17: 1654). Copyright # 2009 John Wiley & Sons, Ltd.

Electrospray ionization (ESI)1–3 and matrix-assisted laser

desorption/ionization (MALDI)4,5 have proven to be

particularly effective in ionizing a wide variety of biological

molecules. For peptides these soft ionization techniques

produce mainly protonated (or multiply protonated) species

in the positive ion mode and collision-induced dissociation

(CID) using tandem mass spectrometric techniques6,7 has

become a widely used method particularly for obtaining the

amino acid identity and sequence in peptides. Under low-

energy CID conditions protonated peptides most often

fragment to produce N-terminal b ions and/or C-terminal

y ions;8–10 indeed, it is the series of such b ions and/or y ions

which provide sequence information. It has been clearly

established11,12 that the y ions are protonated amino acids

(y1) or protonated truncated peptides (ym); however, the

structure(s) of the b ions present a much more complicated

picture. Recent extensive studies13–17 of b2 ions have shown

that, in many cases, cyclization has occurred to form a

protonated five-membered oxazolone ring at the C-terminus

rather than retaining the acylium ion structure initially

proposed.8,9 The majority of evidence points to the bn–ym

mechanism18 for amide bond cleavage and b and y ion

formation, as illustrated in Scheme 1. When there is a strong

nucleophile in the peptide or amino acid side chain,

alternative cyclization reactions involving this nucleophile

may occur.19–22

While the results for b2 ions are consistent with an

oxazolone structure, the first direct experimental evidence

for an oxazolone structure came from elegant infrared

multiphoton dissociation (IRMPD) studies, coupled with

ndence to: A. G. Harrison, Department of Chemistry,y of Toronto, 80 St. George Street, Toronto, ON [email protected] sponsor: Natural Sciences and EngineeringCouncil (Canada).

theoretical calculations, of the b4 ion YGGF derived from

Leu-enkephalin.23,24 The comparison of the observed and

calculated IR spectra clearly indicated the oxazolone

structure. Very recently, two IRMPD studies25,26 have

confirmed the oxazolone structure for b2 ions. The studies

Scheme 1. Mechanism of amide bond cleavage.

Copyright # 2009 John Wiley & Sons, Ltd.

Scheme 2. Fragmentation pathways for b3 ions.

Peptide b3 ions 1299

of the YGGF ion also provided evidence for some population

of a fully cyclic structure for the b4 ion. A number of recent

studies27–34 have shown that larger b ions (b5 and larger) exist

to a significant extent in a fully cyclic form. This cyclic form

may open at different amide bonds resulting in non-

sequence fragment ions on further fragmentation.27–31,34

Earlier studies35,36 also provided evidence for sequence

scrambling.

On the whole peptide b3 ions have seen little study until

lately. An early brief study37 provided results consistent with

an oxazolone structure. While most bn ions with an

oxazolone structure show major fragmentation by loss of

CO to from the respective an ion,13,37,38 b3 ions are unique in

that a3 ions often are not observed in CID mass spectra38–40

although they may be observed in metastable ion mass

spectra of suitable b3 ions.37 Rather fragmentation of b3 ions

results in formation of a3� (a3 – NH3) ions and b2 ions.


Recently, Cooper et al.41 carried out a detailed study of the

formation of the a3� ion from the GGGoxa b3 ion. Contrary to

earlier suggestions38,42 that the ammonia lost contained the

N-terminal amine, they showed that the ammonia lost

contained the nitrogen of the C-terminus residue of the b3

ion. The complex mechanism which they proposed is shown

in modified form in Scheme 2. A more recent study39 has

concluded that the b3 ion has an oxazolone structure which

loses CO to form an a3 ion which is unstable with respect to

fragmentation to the a3� and/or the b2 ion. In agreement with

an earlier computational study,43 no direct b3! b2 fragmen-

tation pathway was found. These detailed studies39,41 of

b3 ionfragmentation haveusedquadrupole iontraptechniques

and, thus, have probed low-energy fragmentation modes. It

appeared desirable to study the evolution of the fragmenta-

tion modes implicit in Scheme 2 with internal energy as well

as to study a wider range of amino acid residues. Such a


DOI: 10.1002/rcm

Figure 1. Breakdown graph for the AAAoxa b3 ion derived

from AAAA.

1300 A. G. Harrison

study is reported here and reveals that the fragmentation

modes of b3 ions depends significantly on the amino acid

residues present and on their positions as well as on the

internal energy of the fragmenting ions. In particular, with

suitable b3 ions, formation of the iminium ion derived from

the C-terminal residue of the b3 ion can become a major

fragmentation reaction. This fragmentation channel is

included in the modified Scheme 2.

Figure 2. Breakdown graph for the YAAoxa b3 ion derived

from YAAAA.

EXPERIMENTAL

All experimental work was carried out using an electrospray

quadrupole time-of-flight (QqToF) mass spectrometer

(QStar, MDS Sciex, Concord, Canada). MS2 experiments

were carried out in the usual fashion for MHþ ions by

selecting the ions of interest with the quadrupole mass

spectrometer Q followed by CID in the quadrupole collision

cell q with mass analysis of the ionic products by the ToF

analyzer. In the MS3 experiments, CID in the interface region

produced fragment ions with those of interest being mass-

selected by the quadrupole Q for fragmentation and analysis

in the usual way. By varying the collision energy in the

collision cell, breakdown graphs, expressing, in a qualitative

way, the relative energy dependencies of the fragmentation

reactions, were obtained under multiple collision conditions.

Ionization was by ESI with the peptide dissolved in

1:1 CH3OH/0.1% aqueous formic acid and introduced into

the source at a flow rate of 80mL min�1. Nitrogen was used as

nebulizing gas and drying gas and as collision gas in the

quadrupole cell.

All peptide samples were obtained from Bachem Bios-

ciences (King of Prussia, PA, USA) and were used as

received.


RESULTS AND DISCUSSION

Figures 1 to 4 present the breakdown graphs for the four b3

ions of nominal structure AAAoxa, YAAoxa, AYAoxa and

AAYoxa. In agreement with earlier work38 the two primary

fragmentation products observed for AAAoxa are the a3� ion

and the b2 ion; specifically no significant signal for the a3 ion

is observed under CID conditions although this product is

observed in metastable ion fragmentation.37 With increasing

collision energy (and, thus, internal energy) formation of

the b2 ion is distinctly favoured over formation of the a3� ion.

In effect, with increasing internal energy the intermediates III

and/or IV of Scheme 2 eliminate the loosely bound

R3CH¼NH (R3¼CH3) moiety to form b2 rather than

continue the rearrangements which eventually lead to

elimination of NH3; such rearrangements are likely to have

an unfavourable entropy of activation. That the b2 ion is

formed rather than the CH3CH¼NHþ2 iminium ion is consis-

tent with the relative proton affinities: PA(CH3CH¼ NH)¼217 kcal mol�1 44 and PA(oxazolone)¼ 222 kcal mol�1.45

These relative proton affinities indicate that the formal

proton transfer reaction IV!V is endothermic in the absence

of hydrogen bonding in the complexes. It appears that such

interactions make the proton transfer feasible. Such was

found to be the case for the GGGoxa system according to the

theoretical calculation of the potential energy surface.41 At

higher internal energies the b2 ion loses CO to form the a2 ion

and the a3� ion loses CO to formm/z 141 (structure unknown).

For both the YAAoxa and AYAoxa b3 ions CID results in

only minor formation of the a3� ion, the major primary

fragmentation reaction being formation of the b2 ion. This ion

fragments further to the a2 ion which, in turn, fragments to

form the tyrosine iminium ion (m/z 136)46 at even higher


DOI: 10.1002/rcm

Figure 3. Breakdown graph for the AYAoxa b3 ion derived

from AYAAA.

Peptide b3 ions 1301

collision energies. The tyrosine residue would be expected to

increase the proton affinity of the oxazolone thus making the

proton transfer reaction IV!V less probable and largely

shutting down the ammonia elimination pathway. The

Figure 4. Breakdown graph for the AAYoxa b3 ion derived

from AAYAA. m/z 136 is tyrosine iminium ion.


increased proton affinity of the oxazolone makes it even less

likely that the iminium ion CH3CH¼NHþ2 would be formed.

The breakdown graph for the AAYoxa b3 ion differs in that

the a3� ion and the tyrosine iminium ion (m/z 136) are the

major primary fragmentation products with only a minor

yield of the b2 ion. It is interesting that no significant a3 ion is

observed even though the tyrosine residue might have been

expected to stabilize the ion. In this system the imine

HOC6H4CH2CH¼NH has a proton affinity (225 kcal mol�1)44

greater than that of the oxazolone (222 kcal mol�1)45 making

the proton transfer reaction IV!V apparently exothermic.

In agreement, we observe substantial formation of the a3� ion

at low internal energies but simple dissociation of the

complex V at higher internal energies with preferential

formation of the tyrosine iminium ion (m/z 136) and only

minor formation of the b2 ion. Unexpectedly, a minor signal

is observed at m/z 235 corresponding to loss of an alanine

residue from the b3 ion and at m/z 207 corresponding to loss

of CO from m/z 235. It is not clear whether this represents a

small extent of full cyclization28,29 of the b3 ion with

reopening to a different sequence or whether the small

extent of cyclization/reopening has occurred for the b4 ion

which fragments extensively to form the b3 ion.

CONCLUSIONS

The present work expands on earlier studies39,41 of the

fragmentation behaviour of peptide b3 ions. The results

obtained support the detailed mechanism proposed by

Cooper et al.41 and expand on this mechanism by including

the fragmentation pathway which yields the iminium ion of

the C-terminal residue of the b3 ion. It might be noted that N-

acetylation eliminates formation of the a� ions,34 consistent

with the mechanism shown in Scheme 2. The present work

and the earlier study41 further illustrate the significant role

ion/neutral complexes play in fragmentation of peptide ions

and the important role relative proton affinities play in

determining the fragment ions which result from decompo-

sition of these complexes.18

AcknowledgementsI am indebted to the Natural Sciences and Engineering

Research Council (Canada) for continued financial support

and to Dr B. Paizs for helpful discussions and for communi-

cation of results prior to publication.

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DOI: 10.1002/rcm

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Fragmentation reactions of some peptide b3 ions: an energy-resolved study