Characterization of α- and γ-glutamyl dipeptides by negative ion collision-induced dissociation

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2004; 39: 136–144Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.515

Characterization of a- and g-glutamyl dipeptides bynegative ion collision-induced dissociation

Alex G. Harrison∗

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada

Received 8 April 2003; Accepted 7 July 2003

The low-energy CID mass spectra of the [M − H]− ions of a variety of dipeptides containing glutamic acidhave been obtained using cone-voltage collisional activation. Dipeptides with the g-linkage, H-Glu(Xxx-OH)-OH, are readily distinguished from those with the a-linkage, H-Glu-Xxx-OH, by the much moreprominent elimination of H-Xxx-OH from the [M − H]− ions of the former isomers, resulting in formationof m/z 128, presumably deprotonated pyroglutamic acid. Dipeptides with the reverse linkage, H-Xxx-Glu-OH, show distinctive fragmentation reactions of the [M − H]− ions including enhanced eliminationof CO2 and formation of deprotonated glutamic acid. Exchange of the labile hydrogens for deuteriumhas shown that there is considerable interchange of C-bonded hydrogens with labile (N- and O-bonded)hydrogens prior to most fragmentation reactions. All dipeptides show loss of H2O from [M − H]−. MS3

studies show that the [M − H − H2O]− ion derived from H-Glu-Gly-OH has the structure of deprotonatedpyroglutamylglycine while the [M − H − H2O]− ions derived from H-Glu(Gly-OH)-OH and H-Gly-Glu-OH show a different fragmentation behaviour indicating distinct structures for the fragment ions.Copyright 2004 John Wiley & Sons, Ltd.

KEYWORDS: ˛-glutamyl dipeptides; �-glutamyl dipeptides; negative ion CID; deuterium labelling; ESI

INTRODUCTION

Tandem mass spectrometry of protonated peptides is widelyused for the determination of the amino acid identities andsequence for the peptide.1 – 4 As a result of many studies themain features of the fragmentation of protonated peptideshave been elucidated, at least at the phenomenologicallevel.5,6 Over the past decade extensive studies haveprovided considerable mechanistic information concerningfragmentation modes and fragment ion structures forprotonated peptides; four recent reviews have summarizedthese studies.7 – 10

On the other hand, rather less is known concerning thedetailed fragmentation modes of deprotonated peptides. Anumber of studies have reported the high-energy collision-induced dissociation (CID) mass spectra of the [M � H]�

ions of mainly di- and tripeptides, where the [M � H]�

ions were produced by fast atom bombardment (FAB).The mechanistic information obtained from these studieshas been summarized by Bowie and co-workers.11,12 Arecent study13 of the low-energy CID of deprotonatedpeptides containing H or alkyl ˛-groups indicated thatmore extensive and sequence-specific fragmentation wasobserved in low-energy CID than in high-energy CID fordeprotonated peptides; this result is consistent with earlier

ŁCorrespondence to: Alex G. Harrison, Department of Chemistry,University of Toronto, 80 St. George Street, Toronto, Ontario M5S3H6, Canada. E-mail: [email protected]/grant sponsor: The Natural Sciences and EngineeringResearch Council (Canada).

studies14 – 17 of non-peptide systems which showed moreextensive and structure-specific fragmentation in low-energyCID of negative ions. Recently, Bowie and co-workers havecarried out extensive studies of the low-energy CID oflarger deprotonated peptides.18 – 24 These studies showed thatuseful sequence information was obtained which often wascomplimentary to that obtained from CID of the protonatedpeptide. This work recently has been reviewed.25 Thesestudies showed that frequently there were fragmentationreactions which were specific to particular ˛-groups in thepeptide. Along the same lines, we recently have shown26

that the benzyl ˛-group has a significant effect on thefragmentation modes of small deprotonated peptides. Thepresent work extends the study of the effect of side-chain functional groups to acidic ˛-groups with a study ofsmall peptides containing glutamic acid. Peptides containingglutamic acid also are of interest since both ˛- and �-linkages are possible and there is considerable interest indistinguishing between the two linkages. In earlier studiesOkada and Kawase27 have examined the electron impactmass spectra of ˛- and �-glutamyl dipeptide methyl estersand showed that the former eliminated an amino acidradical plus CO from the molecular ion while the lattereliminated CH3Ož C CO from the molecular ion. Lloydet al.28 examined the tandem mass spectra of protonated˛-Glu-ε-Lys and �-Glu-ε-Lys produced by FAB ionization.The MHC of the former compound eliminated lysine C COwhile the MHC of the latter eliminated lysine only. Ina recent more extensive low-energy CID study29 it was

Copyright 2004 John Wiley & Sons, Ltd.

CID of deprotonated glutamyl dipeptides 137

shown that protonated dipeptides with an ˛-linkage, H-Glu-Xxx-OH, are characterized by elimination of H2O and byelimination of H-Xxx-OH C CO while protonated dipeptideswith the �-linkage, H-Glu(Xxx-OH)-OH, do not show lossof H2O, but rather eliminate NH3, particularly in metastableion fragmentation, and also eliminate H-Xxx-OH to forman ion of m/z 130. It might be noted that a similarproblem exists in distinguishing between ˛- and ˇ-linkagesin peptides containing aspartic acid and there have beenseveral studies28,30,31 addressing this problem.

EXPERIMENTAL

Collision-induced dissociation studies were carried outusing an electrospray/quadrupole mass spectrometer (VGPlatform, Micromass, Manchester, UK). It is well known32

that CID can be achieved in the interface region betweenthe atmospheric pressure source and the quadrupole massanalyzer and recent work33 – 36 has shown that, by varyingthe field in the interface region in steps, energy-resolvedCID mass spectra can be obtained which are comparable tothose obtained in variable low-energy CID in quadrupolecollision cells. The results of these CID studies are presentedin the following as CID mass spectra at a set cone voltage.Ionization was by electrospray ionization with the sample, atmicromolar concentration in 1 : 1 CH3CN/1% aqueous NH3,introduced into the source at a flow rate of 30 µL min�1.Nitrogen, produced by a W75-72 N2 generator (WhatmanInc, Haverhill, MA, USA), was used as nebulizing gas ata flow rate of 10 L hr�1 and as drying gas at a flow rateof 250 L hr�1. The use of 1 : 1 CD3CN/1% ND3 in D2Oresulted in exchange of all labile hydrogens by deuteriumand formation of the [M � D]� ion in the ionization process;with N2 as the drying gas no evidence for back-exchangein the interface region was observed, although such back-exchange was observed when air was used as the nebulizingand drying gas. MS/MS/MS experiments were carried outusing an electrospray/triple quadrupole mass spectrometer(SCIEX API 3000, Concord, Canada). CID in the interfaceregion produced the fragment ion of interest which wasmass-selected by the first quadrupole mass analyzer andunderwent collisional activation in the r.f.-only quadrupolecollision cell with the ionic fragmentation products beinganalyzed in the final quadrupole mass analyzer.

All peptide samples were obtained from BACHEMBiosciences (King of Prussia, PA, USA). CD3CN (99.8 atom %D) and D2O (99.9 atom % D) were obtained from CambridgeIsotope Laboratories (Andover, MA, USA), whereas theND3OD (26% in D2O, >99 atom % D) was obtained fromCDN Isotopes (Pointe Claire, Quebec, Canada).

RESULTS AND DISCUSSION

There are three isomeric dipeptides which can be formed bycombination of an amino acid H-Xxx-OH with glutamic acid,H-Xxx-Glu-OH, H-Glu-Xxx-OH (˛-linkage) and H-Glu(Xxx-OH)-OH (�-linkage). Bowie and co-workers37 have reportedthe high-energy CID mass spectra of the [M � H]� ions ofa variety of dipeptides with the first two structures but

Table 1. CID spectra for deprotonated H-Glu-Xxx-OH (45 Vcone voltage)

% of base peak

Ionic species Gly Ala Val

-H2 4.6 1.4-H2O 82.7 14.5 7.7-CO2 2.7 1.7 1.3-(H2 C CO2� 16.2 6.8 5.5-(H2O C CO2) 72.3 8.6 12.3-2CO2 27.3 4.5 9.4-(H2O C CO2 C 28) 20.4 5.2 3.1m/z 128 8.2 6.3 6.8y1 100 100 100

did not study any dipeptides with the �-linkage. Theyobserved dominant loss of H2O from the [M � H]� ions withminor formation of m/z 128 (deprotonated pyroglutamicacid?) and minor formation of deprotonated H-Xxx-OH.They concluded that it was difficult to distinguish betweenisomers, the only substantial difference being a greater, butstill minor, loss of CO2 from deprotonated peptides with theH-Xxx-Glu-OH structure.

Table 1 presents the CID mass spectra for the [M � H]�

ions of three dipeptides with structure H-Glu-Xxx-OH, whileFig. 1 compares the spectrum of the [M � H]� ion of H-Glu-Gly-OH with the spectrum obtained for the [M � D]� ionof the peptide in which the labile hydrogens have beenexchanged for deuterium. For deprotonated H-Glu-Gly-OHa prominent fragmentation reaction involves loss of H2O,although this fragmentation mode decreases in importancewhen Gly is replaced by Ala or Val. Figure 1 shows thatloss of water does not involve solely labile hydrogens sinceboth D2O (m/z 187) and HDO (m/z 188) are lost from the[M � D]� ion; this is in agreement with the observations ofBowie and co-workers.37 The [M � H � H2O]� ion fragmentsfurther by elimination of CO2 to give for deprotonated H-Glu-Gly-OH the prominent m/z 141 ion signal. Formationof the y1 ion (deprotonated H-Xxx-OH) provides the basepeak in the CID spectra, particularly for the dipeptidescontaining Ala or Val. The results of Fig. 1 show that they1 ion incorporates, in part, one labile hydrogen and, to agreater extent, two labile hydrogens; similar results wereobtained for the other dipeptides. For dipeptides containingonly H or alkyl ˛-groups the y1 ion has been found13,38

to incorporate only one labile hydrogen; the mechanismproposed by Eckersley et al.38 is presented in Scheme 1.The incorporation of two labile hydrogens suggests anadditional pathway to the y1 ion. A plausible mechanismis presented in Scheme 2 involving participation of the �-carboxyl function. Two further products of note involve lossof H2 C CO2 (m/z 157 in Fig. 1) and loss of 2CO2 (m/z 115in Fig. 1). The latter product cleanly incorporates four labilehydrogens, as expected, while the former incorporates threelabile hydrogens, since HD is lost in the initial step for thelabelled peptide.

The CID mass spectra of the [M � H]� ions of three�-dipeptides H-Glu(Xxx-OH)-OH are presented in Table 2,

Copyright 2004 John Wiley & Sons, Ltd. J. Mass Spectrom. 2004; 39: 136–144

138 A. G. Harrison

Figure 1. Comparison of the CID mass spectrum of deprotonated H-Glu-Gly-OH with the CID mass spectrum of the [M � D]� ion ofthe peptide with the labile hydrogens exchanged for deuterium. Cone voltage: 45 V.

Scheme 1

while Fig. 2 compares the CID spectrum of deprotonatedH-Glu(Gly-OH)-OH with that of the [M � D]� ion of thepeptide in which the labile hydrogens have been exchangedfor deuterium. The major difference from the spectra for the˛-dipeptides (Table 1 and Fig. 1) is the much more prominention signal at m/z 128, most likely deprotonated pyroglutamicacid. Indeed, the y1/m/z 128 ratio can be used to distinguish

Table 2. CID spectra for deprotonated H-Glu(Xxx-OH)-OH(45 V cone voltage)

% of base peak


-H2O 71.0 8.5 5.2-2H2O 5.2 3.1 4.4-(H2O C CO2) 56.9 4.0 6.7-(H2O C CO2 C 28) 15.4 2.5 1.4m/z 128 97.4 54.9 87.3y1 100 100 100

between the ˛- and �-dipeptides, the ratio being in the range12–16 for the former and 1–2 for the latter. The reactionsequence of Scheme 3 can be considered as a plausiblepathway to m/z 128 and to deprotonated H-Xxx-OH(y1 ion). It is unlikely that the amine-deprotonated species isformed directly in the ionization process; rather, it probablyoriginates by proton transfer in an amide-deprotonated orcarboxyl-deprotonated species. The pathways of Scheme 3place one labile hydrogen on the m/z 128 product andtwo labile hydrogens on deprotonated H-Xxx-OH. As thespectra in Fig. 2 show, the actual situation is much morecomplex with the m/z 128 product incorporating, in part,two labile hydrogens and even three to a minor extent,



Figure 2. Comparison of the CID mass spectrum of deprotonated H-Glu(Gly-OH)-OH with the CID mass spectrum of the [M � D]�

ion of the peptide with the labile hydrogens exchanged for deuterium. Cone voltage: 45 V.

Scheme 2

Scheme 3


140 A. G. Harrison

while the y1 ion shows significant incorporation of onlyone and even no labile hydrogens. Evidently, there issignificant interchange of carbon-bonded hydrogens withN- and O-bonded hydrogens prior to fragmentation. Thispresumably involves the hydrogens alpha to the carbonylfunction in the pyroglutamic acid ring since they will bemoderately acidic.

Table 3 presents the CID mass spectra of the [M �H]� ions of three dipeptides H-Xxx-Glu-OH, while Fig. 3compares the spectrum of deprotonated H-Gly-Glu-OH withthat of the [M � D]� ion of the dipeptide with the labilehydrogens exchanged for deuterium. The spectra show amuch greater variety of fragment ions than observed fordeprotonated H-Glu-Xxx-OH or H-Glu(Xxx-OH)-OH. Thereare several distinctive features in the CID mass spectra ofdeprotonated H-Xxx-Glu-OH. These include a much moreintense signal for loss of CO2, formation of the y1 ion(deprotonated glutamic acid, m/z 146), an ion signal at m/z102 and formation of the c1 ion (deprotonated H-Xxx-NH2�.MS3 experiments on the m/z 159 ion �[M � H � CO2]�� ofH-Gly-Glu-OH at 15 eV collision energy showed formationof m/z 141 (H2O loss) 22.6%, formation of m/z 115 (CO2

loss) 31.7%, formation of m/z 102 (loss of the glycineresidue) 31.1%, and formation of the c1 ion (m/z 73) 8.6%.However, the base peak in all spectra is observed to bedeprotonated H-Xxx-OH (m/z 74 in Fig. 3). A plausiblepathway leading to deprotonated H-Xxx-OH is presented

Table 3. CID spectra for deprotonated H-Xxx-Glu-OH (45 Vcone voltage)

% of base peak


-H2 6.8 5.2 9.3-H2O 18.7 17.2 10.9-CO2 35.5 25.7 41.2-(H2 C CO2) 2.0 1.6 2.4-(H2O C CO2) 14.7 10.3 9.6y1(m/z 146) 15.5 3.7 12.2m/z 128 56.6 20.0 47.2-(H2O C CO2 C 28) 12.2 6.9 7.1-2CO2 66.1 26.8 26.9m/z 102 63.8 11.1 23.2[H � Xxx � OH � H]� 100 100 100c1 55.2 30.0 33.4

in Scheme 4 involving the formation of an anhydride as anintermediate. Anhydride intermediates have recently beenproposed by Gronert and co-workers39 and by O’Hair andco-workers40 in the fragmentation of lithiated and protonatedpeptides. Bowie and co-workers37 have previously reportedobservation of deprotonated H-Xxx-OH in the high-energyCID of deprotonated H-Xxx-Glu-OH. The pathway outlined

Figure 3. Comparison of the CID mass spectrum of deprotonated H-Gly-Glu-OH with the CID mass spectrum of the [M � D]� ion ofthe peptide with the labile hydrogens exchanged for deuterium. Cone voltage: 45 V.



Scheme 4

Table 4. MS3 and MS2 studies of ions of m/z 185 (15 eV collision energy)

% of base peak

Ionic species(m/z)

[M � H � H2O]�

Gly-Glu[M � H � H2O]�

�-Glu(Gly)[M � H � H2O]�

˛-Glu-Gly[M � H]�

Pyr-Gly

167 7.8141 21.7 100 100 100128 100123 4.3113 1.8 8.4 9.7 9.885 1.4 1.3 0.684 0.7 0.9 0.682 1.4 1.9 1.274 9.1


142 A. G. Harrison

in Scheme 4 is similar to the pathway they proposed withthe exception that they proposed a concerted rearrangementof the second structure leading directly to the ion/neutralcomplex rather than the stepwise path shown. As indicatedin the scheme, this pathway also could lead to formationof deprotonated pyroglutamic acid (m/z 128). However,another pathway to m/z 128 is revealed by MS3 experimentson the [M � H � H2O]� ion derived from H-Gly-Glu-OHwhere m/z 128, formed by elimination of the glycineresidue is a major product (Table 4). The exchange oflabile hydrogens for deuterium (Fig. 3) shows that there is

considerable interchange of labile hydrogens with C-bondedhydrogens prior to formation of the m/z 128 product.

Table 4 compares the CID mass spectra (obtained byMS3 experiments) of the [M � H � H2O]� (m/z 185) ionsderived from H-Gly-Glu-OH, H-Glu(Gly-OH)-OH and H-Glu-Gly-OH with the CID spectrum of deprotonated pyrog-lutamylglycine (H-Pyr-Gly-OH). The good agreement of thespectrum of the m/z 185 ion from H-Glu-Gly-OH with thatfor deprotonated H-Pyr-Gly-OH is strong evidence thatloss of water from deprotonated H-Glu-Gly-OH resultsin cyclization to the pyroglutamyl derivative. The m/z

Figure 4. CID mass spectra of the [M � H]� ions of H-Glu-Tyr-OH, H-Glu(Tyr-OH)-OH and H-Tyr-Glu-OH. Cone voltage: 45 V.



185 ion derived from H-Glu(Gly-OH)-OH, while showingsubstantial loss of CO2, does show a distinctive m/z 167(-H2O) fragment indicating that at least some of the m/z185 ions have a structure distinct from that of deproto-nated H-Pyr-Gly-OH. The H-Glu(Xxx-OH)-OH peptides arethe only isomers which show (Table 2) loss of two watermolecules in the CID mass spectra of the [M � H]� ions.The m/z 185 ion derived from H-Gly-Glu-OH gives a verydistinctive CID mass spectrum and it is probable that, inthis case, deprotonated H-Gly-Pyr-OH is formed. Bowie andco-workers37 have previously noted that high-energy CID ofthe [M � H � H2O]� ions derived from H-Ala-Glu-OH andH-Glu-Ala-OH results in distinctly different spectra.

The results in Tables 1–3 show that it is possible todistinguish isomeric dipeptides containing glutamic acid bylow-energy CID of the [M � H]� ions. The major distinctionof the ˛-dipeptide, H-Glu-Xxx-OH, from the �-dipeptide, H-Glu(Xxx-OH)-OH, is the much more pronounced formationof m/z 128 (deprotonated pyroglutamic acid?) for thelatter. The dipeptides with the Xxx-Glu-OH structure arecharacterized by the much more pronounced loss of CO2

from [M � H]� and by formation of the y1 ion, deprotonatedglutamic acid (m/z 146). These differences carry over to morecomplex dipeptides, as illustrated by the CID mass spectraof the [M � H]� ions of the three dipeptides containingtyrosine and glutamic acid presented in Fig. 4. Althoughdeprotonated tyrosine (m/z 180) is prominent in all spectrathe �-dipeptide is characterized by the intense m/z 128 ionsignal while the H-Tyr-Glu-OH dipeptide is characterizedby the more intense [M � H � CO2]� (m/z 265) ion signaland the ion signal at m/z 146 corresponding to deprotonatedglutamic acid. All three spectra show a weak ion signal atm/z 185 corresponding to elimination of OC6H4CH2 (106Da) from the [M � H � H2O]� ion. Bowie and co-workers41

have previously noted the loss of 106 Da from deprotonatedpeptides containing tyrosine. The one system studied wheredistinction of the ˛- and �-dipeptides was not possible wasfor the pair H-Glu-Glu-OH and H-Glu(Glu-OH)-OH wherethe [M � H]� ions of both gave m/z 128 (deprotonatedpyroglutamic acid?) and deprotonated glutamic acid (m/z146) as the major fragmentation products with similarrelative abundances for the two isomers.

CONCLUSIONS

Isomeric dipeptides containing glutamic acid with the ˛-linkage, H-Glu-Xxx-OH, or the �-linkage, H-Glu(Xxx-OH)-OH, can be readily distinguished by low-energy CID ofthe [M � H]� ions, since those with the �-linkage showmuch more pronounced elimination of H-Xxx-OH to formm/z 128, presumably deprotonated pyroglutamic acid. Thethird isomers, H-Xxx-Glu-OH, show distinctive fragmentions arising from [M � H]� including enhanced loss of CO2

and formation of deprotonated glutamic acid. In agreementwith earlier studies13,26 of deprotonated peptides, low-energycollisional activation leads to more extensive structure-specific fragmentation than does high-energy collisionalactivation.

Finally, while plausible fragmentation mechanisms havebeen proposed above, the substantial interchange of labile

and C-bonded hydrogens observed indicates that thedetailed fragmentation mechanisms are more complex thanpictured in the schemes.

AcknowledgementsThe continued financial support of the Natural Sciences andEngineering Research Council (Canada) is gratefully acknowledged.Professor K. W. M. Siu (Centre for Research in Mass Spectrometry,York University) kindly provided access to the triple quadrupolemass spectrometer. I thank Houssain El Aribi for his assistance withthe MS3 experiments.

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Characterization of α- and γ-glutamyl dipeptides by negative ion collision-induced dissociation