Fragmentation in MSMS

FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES

Bela Paizs1,2* and Sandor Suhai11Department of Molecular Biophysics, German Cancer Research Center,Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany2Protein Analysis Facility, German Cancer Research Center,Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany

Received 17 July 2003; received (revised) 29 February 2004; accepted 5 March 2004

Published online 12 July 2004 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20024

The fragmentation pathways of protonated peptides are reviewedin the present paper paying special attention to classification ofthe known fragmentation channels into a simple hierarchydefined according to the chemistry involved. It is shown that the‘mobile proton’ model of peptide fragmentation can be used tounderstand the MS/MS spectra of protonated peptides only in aqualitative manner rationalizing differences observed for low-energy collision induced dissociation of peptide ions having orlacking a mobile proton. To overcome this limitation, a deeperunderstanding of the dissociation chemistry of protonatedpeptides is needed. To this end use of the ‘pathways incompetition’ (PIC) model that involves a detailed energeticand kinetic characterization of the major peptide fragmentationpathways (PFPs) is proposed. The known PFPs are described indetail including all the pre-dissociation, dissociation, and post-dissociation events. It is our hope that studies to further extendPIC will lead to semi-quantative understanding of the MS/MSspectra of protonated peptides which could be used to developrefined bioinformatics algorithms for MS/MS based proteomics.Experimental and computational data on the fragmentation ofprotonated peptides are reevaluated from the point of view of thePIC model considering the mechanism, energetics, and kineticsof the major PFPs. Evidence proving semi-quantitative predict-ability of some of the ion intensity relationships (IIRs) of the MS/MS spectra of protonated peptides is presented.# 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:508–548,2005Keywords: protonated peptides; peptide fragmentation; reac-tion mechanism; mass spectrometry

I. INTRODUCTION

A. Protein Sequencing by Mass Spectrometry (MS)

MS has become an important tool for determining the amino acidsequence of peptides and proteins. The MS technique involvescreation and detection of charged peptide and protein ions in thegas phase. Soft ionization techniques like electrospray ionization(ESI, Fenn et al., 1989) and matrix-assisted laser desorption/ionization (MALDI, Karas & Hillenkamp, 1988) are used toproduce intact peptide and protein ions in the gas phase (mostly in

positive ion mode, e.g., via protonation) without fragmentation.The mass to charge ratio (m/z) of these ions can be rapidly andaccurately measured allowing such applications like fast evalua-tion of the correctness of the sequence of peptides and proteins,checking the presence of post-translational modifications, andapplication of bioinformatics-assisted peptide-mass fingerprint-ing (PMF, Henzel et al., 1993; James et al., 1993; Mann, Hojrup,& Roepstorff, 1993; Pappin, Hojrup, & Bleasby, 1993; Yateset al., 1993) methods. Analysis by using PMF is based ondigestion of the protein with a site-specific enzyme (mostlytrypsin) and comparison of the measured peptide molecularweights (peptide mass fingerprint) to those predicted in silico forthe sequences in protein and/or translated nucleic acid databases.Provided that a sufficient number of peptide ions are observed inthe MS experiment and the protein is not heavily modified, amatch can be generally found.

The power of this bioinformatics based strategy can bedramatically increased by employing methods of tandem massspectroscopy (McLafferty, 1983). In the tandem MS (MS/MS)and MSn experiments, the first mass analyzer is used toselectively pass an ion into another reaction region whereexcitation and dissociation take place. The second mass analyzeris used to record them/z values of the dissociation products. (MS/MS experiments in the quadrupole ion trap or Fourier transformion cyclotron instruments are tandem in time and use the samevolume for the above processes.) Excitation of the precursor ionis most commonly achieved by energetic collisions with a non-reactive gas, such as argon or helium, and is referred to ascollision-induced (activated) dissociation (CID or CAD).

The observed fragmentation pattern depends on variousparameters including the amino acid composition and size of thepeptide, excitation method, time scale of the instrument, thecharge state of the ion, etc. Peptide precursor ions dissociatedunder the most usual low-energy collision conditions fragmentalong the backbone at the amide bonds (Hunt et al., 1986;Biemann, 1988; Papayannopoulos, 1995) forming structurallyinformative sequence ions and less useful non-sequence ions bylosing small neutrals like water, ammonia, etc. The sequence ionsinvolve b and y ions, which contain the N- and C-terminus,respectively. (For the nomenclature (Roepstorff & Fohlmann,1984; Biemann, 1988) applied, see Scheme 1.)

The information available in the MS/MS spectra ofprotonated peptides can be used to identify proteins in severalways. Amino acid residues can be determined from the massdifference of successive fragment ions of the same type (e.g., bn

and bn� 1). In this way, one can apply tandem MS for evende novo

Mass Spectrometry Reviews, 2005, 24, 508– 548# 2004 by Wiley Periodicals, Inc.

————*Correspondence to: Bela Paizs, Protein Analysis Facility, Deutsches

Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidel-

berg, Germany. E-mail: [email protected]

peptide sequencing (Dancik et al., 1999; Taylor & Johnson, 2001)provided the corresponding ion series are present in the MS/MSspectrum. Some algorithms make use of MS/MS data to generate‘peptide-sequence tags’ (Mann & Wilm, 1994) that consist ofinformation on a short stretch of sequence. Another approach isbased on in silico generation of MS/MS fragmentation patterns(Eng, McCormack, & Yates, 1994; Clauser, Baker, & Burlin-game, 1999; Perkins et al., 1999) for peptides derived fromentries of protein and translated nucleic acid databases andcomparison of the predicted spectra to those determinedexperimentally.

It is evident from this short introduction that the level of ourunderstanding of the main fragmentation processes of protonatedpeptides is critical not only from the academic point of view butalso for practical reasons. For example, deriving ‘peptide-sequence tags’ from the MS/MS spectra requires some knowl-edge of the major fragmentation pathways and rules. Sinceprotonated peptides dissociate on a large number of differentfragmentation pathways, generation of in silico MS/MS spectraconsidering both the ion m/z and fragment ion abundancedimensions for general peptide entries of databases is an ex-tremely difficult task. Also, some peptides show selective and/orenhanced fragmentation (Wysocki et al., 2000) at some of theamino acid residues producing MS/MS spectra poor in valuablesequence ions. In the light of these facts, it is not surprising thatexisting sequencing programs use only the information inherentin them/z values of the most important sequence ions and discardany ion intensity-related data. Protein identification, usingtandem MS could be no doubt further developed if the majorrules deriving the fragmentation of protonated peptides wereknown to such an extent that would permit predictions of some ofthe fragment ion intensity relationship (IIRs) of the MS/MSspectra of protonated peptides.

There are two major ways to determine IIRs for the MS/MSspectra of protonated peptides. The first, a ‘top down’ strategypioneered by Wysocki, Yates, and Simpson (Huang et al., 2002;Kapp et al., 2003; Tabb et al., 2003) is a statistical approach basedon systematic assessment of large databases containing MS/MSspectra of protonated peptides to derive fragmentation rules. Thesecond, a ‘bottom up’ chemical approach involves systematicinvestigation of the major fragmentation pathways of protonatedpeptides to increase our knowledge on the dissociation chemistryinvolved. The personal view of the present authors is that newhighly efficient peptide sequencing algorithms utilizing MS/MS

IIRs for protonated peptides will be based on results delivered byintensive interplay of the ‘top down’ statistical and the ‘bottomup’ chemical approaches.

The present paper reviews the dissociation chemistry ofprotonated peptides by paying special attention to recentdevelopments that could be used to explain and in some extentto predict MS/MS IIRs.

B. Mobile Proton Model

The most comprehensive model currently available to describehow protonated peptides dissociate upon excitation is termed the‘mobile proton’ model. This has emerged as a result of a largenumber of studies performed by Wysocki (Jones et al., 1994;Dongre et al., 1996; Tsaprailis et al., 1999; Wysocki et al., 2000),Harrison (Tsang & Harrison, 1976; Harrison & Yalcin, 1997),Gaskell (Burlet, Yang, & Kaskell, 1992; Cox et al., 1996;Summerfield, Whiting, & Gaskell, 1997; Summerfield, Cox, &Gaskell, 1997), Boyd (Tang & Boyd, 1992; Tang, Thibault, &Boyd, 1993), and others.

Protonated peptides activated under low-energy collisionconditions fragment mainly by charge directed reactions(Johnson, Martin, & Bienmann, 1988; Burlet, Yang, & Kaskell,1992; Tang & Boyd, 1992; McCormack et al., 1993; Tang,Thibault, & Boyd, 1993; Somogyi, Wysocki, & Mayer, 1994;Cox et al., 1996). Being multifunctional compounds, peptidescan be protonated at various protonation sites (terminal aminogroup, amide oxygens and nitrogens, side chain groups) leadingto various isomers. There are two major classes of peptide ions,which differ in the energetics of the isomers produced byprotonation. In the first class, one or more of the protonation sitesis energetically and/or kinetically more favored than the othersleading to sequestration of the added proton(s), for example,singly protonated tryptic peptides containing arginine at the C-terminus. Characteristic of this peptide ion class is the largeenergy that is needed to mobilize the extra proton(s) toenergetically less favored protonation sites. For the secondgroup, many of the protonation sites are accessible in a narrowenergy range. This class is best represented by the practicallyimportant doubly charged tryptic peptides.

As the internal energy of the ions increases upon excitation,energetically less favored protonation sites like those of thereactive intermediates leading to backbone dissociation canbecome more populated or in the case of hard proton se-

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questering, charge-remote fragmentation (e.g., ‘aspartic acid’effect, see later) can occur. Considering the reactive intermedi-ates of the backbone fragmentation, it is known from the resultsof molecular orbital calculations (McCormack et al., 1993;Somogyi, Wysocki, & Mayer, 1994) that protonation on theamide nitrogen leads to considerable weakening of the amidebond, whereas protonation on the amide oxygen makes the amidebonds even stronger than those in the neutral species. On the otherhand, protonation on the amide nitrogen is not thermodynami-cally favored compared to protonation on amide oxygens, or ontheN-terminal amino group or basic amino acid (AA) side chainssuch as those of arginine and lysine. In short, from the point ofview of decomposition, protonation on the amide nitrogen isfavorable, whereas from the thermodynamic point of view thissite is not the most favored one. The ‘mobile proton model’introduced by Wysocki (Dongre et al., 1996) and co-workersresolves this discrepancy by stating that upon excitation theproton(s) added to a peptide will migrate to various protonationsites prior to fragmentation provided they are not sequestered bya basic amino acid side chain. Essentially the same concept hasbeen developed in Gaskell’s group (Burlet, Yang, & Kaskell,1992) by introducing the ‘heterogeneous population model’ thatassumes the existence of different protonated forms for easilyfragmenting species and the dominance of a single structure forthose peptide ions in which the added proton is sequestered.

The ‘mobile proton’ model has been verified by usingdeuterium labeling techniques (Tsang & Harrison, 1976;Mueller, Eckersley, & Richter, 1988; Johnson, Krylov, & Walsh,1995; Harrison & Yalcin, 1997) which indicate strong H/Dmixing prior to collisionally activated dissociation of [MþD]þ

ions of a number of amino acids and small peptides. In a veryearly study, Tsang & Harrison (1976) have shown that facile H/Dmixing occurs in the D2 and CD4 chemical ionization of aminoacids. This work was later extended (Harrison & Yalcin, 1997) tosmall peptides lacking arginine, the [MþD]þ ions of whichshow high proton mobility between the terminal amino group andamide Ns and Os. Mueller, Eckersley, & Richter (1988) haveinvestigated the fragmentation of the [MþH]þand [MþD]þ

ions of H-Phe–Phe–Phe-OH and found that practically completeexchange of the added deuteron with labile hydrogens occur uponexcitation. Johnson, Krylov, & Walsh (1995) have studied thefragmentation reactions of singly deuterated peptides andhave found that the deuteron is redistributed amongst theexchangeable sites upon excitation to induce fragmentation ofthe parent ion.

Wysocki and co-workers have demonstrated (Jones et al.,1994; Dongre et al., 1996; Wysocki et al., 2000) that the relativepositions of fragmentation efficiency curves obtained by ESI incombination with surface induced dissociation (ESI/SID) dependon the amino acid composition (absence or presence and type of abasic residue) and on the sequence and the size of the peptideinvestigated. These studies have benefited from the relativelynarrow internal energy distribution of SID-generated ions and thefact that the average energy of the ion population can be easilychanged. Investigation of a large number of peptides withsystematically changed amino acid composition showed thatbackbone dissociation of protonated peptides under low-energyconditions is a charge-directed process. Also, the energy requiredfor proton ‘mobilization’ from a basic side chain or the amino-

terminus depends on the amino acid composition, with dissocia-tion energy requirements greatest for arginine-containing pep-tides and decreasing in the order of Arg-containing>Lys-containing> non-basic peptides, mimicking the order ofdecreasing gas-phase basicity. In selected cases, more energymight be required to mobilize the sequestered proton than isrequired to initiate charge-remote fragmentation pathways(aspartic acid effect, see later). Mechanistic considerationsinvolved in the ‘mobile proton model’ have been refined by theWysocki and Gaskell groups (Tsaprailis et al., 1999) to such anextent that allows qualitative understanding of the fragmentationbehavior of peptides containing a few arginines and/or acidicresidues and/or protons. Also, the ‘mobile proton’ model wassuccessfully applied to explain the charge state dependentfragmentation of gaseous protein ions (Engel et al., 2002). Inthose cases, when the number of ionizing protons is larger thanthe number of arginines, non-selective fragmentation is observ-ed. If the number of arginines is larger or equal to the number ofionizing protons, selective fragmentation via the aspartic acideffect is expected.

The ‘mobile proton’ model has also been validated by usingtheoretical tools (Csonka et al., 2000, 2001; Paizs et al., 2001).Using quantum chemical techniques, the structure and energeticsof various protonated forms of model peptides were determined.These species are connected by transitions like internal rotations,proton transfer reactions, etc. After determining the structuresand energetics of the corresponding transition structures, one canapply quantum chemical data (like relative energies andvibrational frequencies) in the RRKM formalism to approximatethe unimolecular reaction rates and the time scale of theunderlying processes. By collecting all these data, it is possibleto construct ‘proton traffic maps’ (Csonka et al., 2000) thatcontain the energy required to mobilize the added proton betweenvarious protonation sites including the terminal amino group,amide Ns and Os, and amino acid side chains. These theoreticalinvestigations unequivocally proved that the energy require-ments for mobilizing the added proton increase in the N-formyl-glycineamide (no terminal amino group, transitions only be-tween amide oxygens and nitrogens, Csonka et al., 2000), H-Gly–Gly-OH (Paizs et al., 2001), and H-Lys–Gly-OH (Csonkaet al., 2001) series in agreement with the ‘mobile proton’ model.For the investigated systems, it was shown that the added protoncan sample all protonation sites prior to fragmentation at internalenergies well below the threshold energy of the most favoredfragmentation pathway.

C. Peptide Fragmentation Pathways (PFPs):the ‘Pathways in Competition’ (PIC)Model of Peptide Dissociation

The appearance of a particular sequence ion in the MS/MSspectra of protonated peptides depends on two major factorswhich include the probability for the cleavage of the correspond-ing amide bond and mechanistic aspects that decide which frag-ment will keep the added proton(s) during the spatial separationof the products. The dissociation probability depends on theenergetic and kinetic accessibility of the reactive configurationsand on the actual rate constants of the bond cleavages themselves.

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In most of the cases, the fate of the separating fragments isdecided by the thermodynamics involved, that is, the fragmentwith larger proton affinity (PA) will usually keep the added protonresponsible for the charge-directed dissociation. In other words,formation of fragment ions of protonated peptides involves pre-dissociation (proton transfer reactions, transitions betweenisomers and tautomers, cis– trans isomerization of amide bonds,etc.), dissociation, and post-dissociation events. One can fullyunderstand the MS/MS spectra of protonated peptides only if themajor mechanistic, energetic, and kinetic aspects of the pre-dissociation, dissociation, and post-dissociation processes areknown.

In the following, we briefly evaluate the ‘mobile protonmodel’ of peptide fragmentation considering the very generalpoints described in the previous paragraph. Following theintroduction of ‘soft’ ionization techniques like ESI and MALDI(Karas & Hillenkamp, 1988; Fenn et al., 1989) and the generalavailability of triple quadrupole and ion trap instruments, the late1980s and early 1990s saw an unambiguous shift in MS/MSpeptide analysis from the high to the low fragmentation-energyregime. The high-energy MS/MS spectra of protonated peptidescontain ions formed on both backbone and charge-remotefragmentation pathways (Papayannopoulos, 1995). The latterreactions do not correlate with the position of the ionizing protonon the peptide chain. The majority of the high-energyfragmentation pathways are initiated by reactions involvingdirect bond cleavages. Such reactions are known to be facile onlyif the ion population is significantly excited. Changing theexcitation conditions to favor the low fragmentation-energyregime made the high-energy charge-remote dissociationchannels inaccessible since the majority of the ions excitedunder low-energy collision conditions do not have enough energyto fragment in reactions involving direct bond cleavages atobservable rates. However, protonated peptides have proved todissociate in the low fragmentation-energy regime efficiently.The major success of the ‘mobile proton model’ was to provide asolid background to qualitatively understand the underlyingchemistry (i) by proving direct involvement of the ionizingproton in the majority of the low-energy fragmentation processes(charge-directed pathways), (ii) by showing the lability ofnitrogen protonated amide bonds, and (iii) by explaining howselective charge-remote processes (e.g., aspartic acid effect, seebelow) can become competitive if the mobility of the addedproton is dramatically decreased by sequestration of stronglybasic amino acid side chains.

Contrary to the many successful attempts used to qualita-tively explain the MS/MS spectra of various protonated peptides,the ‘mobile proton’ model considers only pre-dissociationprocesses of peptide fragmentation discussing mainly accessi-bility or inactivity of proton-transfer pathways which lead toreactive intermediates of sequence ion fragmentation channels.For example, making a distinction between active and inactiveproton transfer pathways is enough to explain why doublycharged tryptic peptides containing Arg at the C-terminusfragment more easily than the corresponding singly chargedspecies. However, the ‘mobile proton’ model is not able to answersuch simple questions, as to why the low-energy CID spectra ofprotonated Leu-enkephaline is dominated by loss of the C-terminal Leu residue or why many of the fragment ions expected

to be present in the MS/MS spectra of doubly charged trypticpeptides do not appear at all.

As mentioned above clear understanding of the fragmenta-tion pathways and the key factors determining the MS/MS ionabundances of protonated peptides is needed to improve theexisting bioinformatics based protein identification tools. Overthe past years, there has been considerable activity to providemore detailed information on the ion structures and fragmenta-tion pathways of protonated peptides. However, both the ‘bottomup’ chemical and ‘top down’ statistical approaches are still intheir infancy. The first statistical evaluations of the MS/MS dataof a large number of protonated peptides are just appearing(Huang et al., 2002; Kapp et al., 2003; Tabb et al., 2003) and thereis still a lot to understand in the dissociation chemistry ofpeptides. There is also no doubt that the ‘bottom up’ chemicalstrategy has to go far beyond the ‘mobile proton’ model whichdescribes only pre-dissociation events of peptide fragmentation.A new—more powerful—model is needed which could be usedto predict at least some of the IIRs of the MS/MS spectra ofprotonated peptides. To do so, the new model has to involve atleast a semi-quantitative description of all pre-dissociation,dissociation, and post-dissociation processes of the mostimportant fragmentation pathways.

In the following, we outline the major characteristics of thepathways in competition (PIC) model of peptide fragmentationby analyzing the dissociation chemistry involved in the differentfragmentation pathways. This model is based on classification ofthe PFPs into a simple hierarchy; mechanistic, energetic, andkinetic description of the individual pathways; and a kineticapproach used to describe competition of the various pathways. Itis to be noted here that the PIC model summarizes the results of alarge number of studies performed in the Boyd, Bursey, Harrison,Gaskell, Glish, O’Hair, Paizs, Vaisar, Wesdemiotis, Wysocki, andco-workers. We do believe that in the near future PIC will providea flexible, concise, and powerful framework that will be used inthe ‘top down’ statistical approach as a data model to determineMS/MS IIRs for improved protein identification tools.

1. Pathways in Competition Modelof Peptide Fragmentation

Dissociation of protonated peptides can be described as acompetition between charge-remote and charge-directed PFPs ina complicated reaction pattern where fragment ions are formedwith substantially different probabilities. PFPs can be classifiedaccording to a hierarchy shown in Scheme 2. Protonated peptidesin the low fragmentation-energy regime dissociate mainly oncharge-directed pathways. The only low-energy charge-remotePFPs correspond to the selective cleavage observed for some ofthe Asp containing peptides (aspartic acid effect, for details seebelow) and side chain reactions of oxidized methionine. Thecharge-directed PFPs can be further classified as sequence ornon-sequence dissociation channels. The former produce ionscontaining information on the primary structure of peptideswhereas the latter correspond to losses of small neutrals likewater, ammonia, etc. The structurally most valuable b and yfragment ions of protonated peptides are primarily formed onsequence PFPs by cleavage of the amide bond. The PFPs leading


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to b and y ions involve migration of the added proton from theenergetically most preferred protonation site to amide nitrogens.Protonation of the amide nitrogen has two profound effects: (1) itweakens the amide bond; (2) the carbon atom of the protonatedamide group becomes a likely target of a nucleophilic attack ofnearby electron-rich groups.

Amide nitrogen protonated species can dissociate by directbond cleavage, for example, for the N-terminal amide bond ofunderivatized protonated peptides on the a1� yx pathway. How-ever, the low fragmentation-energy regime disfavors dissociationvia direct bond cleavage because these reactions are facile only iflarge energy is deposited into the ions. Therefore, the majority ofthe amide bonds are cleaved in more complex rearrangement-type reactions involving nucleophilic attack of various function-alities on the carbon center of the protonated amide bon. Thesefunctionalities involve either (i) the oxygen of the N-terminalneighbor amide bond (bx� yz pathway) or (ii) the nitrogen of theN-terminal amino group (aziridinone and diketopiperazinepathways) or (iii) side chain nucleophiles. The b and y ionsformed on the primary sequence PFPs can fragment further toform lower b ions (bx! bx� 1 pathway), a ions (bx! ax

pathway), internal fragments, and internal immonium ions.Competition of these pathways is one of the major factorsdetermining the MS/MS spectra of protonated peptides.

For all the low-energy PFPs described above, the fate of theseparating fragments is decided by the thermodynamics involved,that is, the fragment with larger PA will usually keep the addedproton. The only exceptions are those cases where the fragmentationintroduces chemical changes, which lead to a fixed charge (e.g., seediscussion below on dissociation of peptides containing N-methylated amide bonds). It is worth noting here, that empiricalformulas have already been derived for some of the PFPs (bx� yz

and a1� yx) which allow prediction of some MS/MS IIRsconsidering the thermodynamics of the separation of the fragments.

Some IIRs can be derived from analysis of individual PFPs(Paizs & Suhai, 2002b; and see discussion of the bx� yz

pathways below) if the MS/MS spectrum is dominated by thecorresponding dissociation channels. However, a more completeunderstanding of the MS/MS spectra of protonated peptidesrequires the introduction and integration of kinetic models of theunderlying complex reaction pattern.

In the following, experimental and theoretical tools used tocharacterize PFPs are briefly discussed. In the main body of thepaper, the chemistries of the various PFPs classified in thehierarchy of Scheme 2 are critically reviewed.

D. Experimental Tools for Investigating PeptideFragmentation Pathways

The experimental tools that can be applied to probe the mecha-nistic, energetic, and kinetic details of PFPs involve well-established techniques of MS like MSn experiments, comparisonof the MS spectra of fragment ions with those of well-definedreference compounds synthesized independently, exploring theneutrals co-produced with fragment ions, isotope labeling,blocking of possible reaction sites, etc. (O’Hair, 2000; Polce,Ren, & Wesdemiotis, 2000; Wysocki et al., 2000). We do notdescribe these techniques here; the reader is referred to thediscussion of the various PFPs for possible applications.

A few techniques can provide especially useful informationon the energetics and kinetics of PFPs. Morgan & Bursey (1994,1995) and Harrison and co-workers (Harrison et al., 2000;Harrison, 2002) studied the relationship between various ionabundances and the PA of the constituting amino acids in series ofprotonated di- and tri-peptides. For some of the peptides seriesinvestigated, a linear free energy relationship was foundindicating a direct dependence between thermochemistry andthe kinetically determined logarithms of relative ion abundances.

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Energy-resolved CID and SID studies can be used to probeenergetic details of the PFPs. Klassen & Kebarle (1997) appliedenergy-resolved CID-MS to determine the dissociation thresholdenergies of fragment ions of various protonated peptides. Theenergetics and the dynamics of the primary fragmentationpathways of small peptides were determined from the RRKMmodeling of the collision energy-resolved fragmentation effi-ciency curves (Laskin, Denisov, & Futrell, 2000). Hanley and co-workers (Lim et al., 1999) developed a method for determinationof dissociation energies using SID techniques.

Blackbody infrared radiative dissociation (BIRD) devel-oped by Price, Schnier, & Williams (1996) and Dunbar &McMahon (1998) can also be used to obtain activation energiesand dynamics of dissociation processes of medium-sized andlarge biomolecule ions. With BIRD, trapped ions are activated byinteraction with the blackbody radiation field inside the vacuumchamber of a Fourier-transform mass spectrometer at very lowpressures. From the temperature dependence of the unimolecularrate constants, Arrhenius activation parameters can be obtained(Schnier et al., 1997).

E. Theoretical Tools for Investigating PeptideFragmentation Pathways

Studying the energetics and kinetics of PFPs by means oftheoretical methods requires special modeling strategies andinvolves scanning of the potential energy surface (PES) ofprotonated peptides, determining transition structures belongingto pre-dissociation and dissociation events and the determinationof thermochemical quantities (proton affinities) of final products.

Scanning the PES of protonated peptides is important toprobe the energetics of the different protonations sites, chargesolvated and salt-bridge (SB) forms, etc. (Csonka et al., 2000;Paizs et al., 2002). A generally used computational strategy toobtain low-energy structures of peptides is based on moleculardynamics simulations using protein force fields. Direct applica-tion of the available modeling strategies for the case ofprotonated peptides is not straightforward since some of theatom types (protonated amide nitrogen and oxygen) are missingfrom the protein force fields used nowadays. This shortcomingcan be circumvented by running the dynamics calculations onneutral species and protonating a collection of neutral structuresin the second step. The disadvantage of this method is that a largenumber of such protonated species have to be further refined asneutral and protonated species can differ significantly. A betterapproach is to derive the missing parameters and run thedynamics calculations using the extended protein force fields.Our laboratory is currently involved in a project aimed to defineparameters for protonated amide nitrogen and oxygen atoms. Ourexperience shows that even the best structures derived fromdynamics runs have to be refined by quantum chemicalcalculations which should be performed at various levels startingwith less accurate models and finishing with density functionaltheory (DFT) computations on the most interesting species.

Since MS/MS ion abundances are determined by the kineticsof the PFPs, transition structures belonging to the investigatedfragmentation pathways, proton transfer reactions, intramolecu-lar rearrangements, etc. have to be searched for. It is worth notinghere that one can obtain reasonable results using only moderate

basis sets in conjunction with DFT for the chemistry ofprotonated peptides. This is mainly because of the facts that thepositive charge of the ion forces the electrons close to the nucleiand the most important chemical changes are heterolytic bondcleavages, which can be described at the DFT levels reasonably.Efficient data handling in the PES scan phase of the modeling andthe relative ‘simplicity’ of the chemistry involved, allowresearchers to perform detailed studies on protonated oligopep-tides having up to 6–7 amino acid residues.

Using the results of the quantum chemical calculations, therate coefficients for the transitions between the minima on thePES can be calculated using the RRKM method (Forst, 1973;Holbrook, Pilling, & Robertson, 1996). Our experience showsthat the RRKM calculations are particularly useful to predict thetime-scale of the various processes occurring for protonatedpeptides because the currently used mass spectrometers cover anextremely wide range (10�9–103 sec) of reaction times.

F. Nomenclature

Throughout the present paper, we refer to fragment ions ofprotonated peptides using the nomenclature (Scheme 1) devel-oped by Roepstorff & Fohlmann (1984) and modified byBiemann (1988). The dissociation mechanisms sometimesdepend on the location of the cleaved amide bond along thepeptide backbone. Various amide bonds are denoted byspecifying the amino acid (aa) residues connected by the amidebond, that is, aa(1)–aa(2), aa(2)–aa(3), . . . , aa(n)–aa(nþ 1) referto the N-terminal first, second, . . . ,nth amide bond in a generalaa(1)–aa(2)–aa(3) . . . aa(n) . . . peptide (containing N amino acidresidues), respectively. Amino acid residues are denoted by theirthree-letter code. Some of the PFPs are referred as specific ornon-specific in the text. By the former, we note pathwaysinvolving chemistry determined by a particular amino acid sidechain moiety whereas the later is determined purely by theinteraction of backbone atoms.

II. CHARGE-DIRECTED PEPTIDEFRAGMENTATION PATHWAYS

A. Dissociation of the aa(n)–aa(n+1) (n>1) AmideBond of Protonated Peptides

According to the general scheme described in Introduction of thepresent article, protonated amide bonds can be cleaved either byrearrangement-type reactions or by direct bond cleavage. Underlow-energy collision conditions most of the backbone cleavages ofprotonated peptides occur in reactions which belong to the formerclass (the only known exception is the a1� yx pathway discussedbelow). This is mainly because the excited ions do not have enoughenergy to fragment on direct bond cleavage pathways, which arefacile, only if significant energy is deposited into the ions. It is,therefore, not surprising that the majority of the sequence ions ofprotonated peptides are formed on unspecific fragmentationpathways where either the N-terminal neighbor amide oxygen(bx� yz pathway, Paizs & Suhai, 2002a) or the nitrogen of theN-terminal amino group (diketopiperazine pathway, Cordero,


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Houser, & Wesdemiotis, 1993) attacks the carbon center of theprotonated amide bond to induce dissociation.

1. bx� yz Peptide Fragmentation Pathways

The major steps of the bx� yz pathway are depicted in Scheme 3afor a general peptide. The bx� yz pathway is initiated bymobilization of the added proton (in Scheme 3a it is located at the

N-terminal amino group; some amino acid side chains might bemore preferred) to the nitrogen of the amide bond to be cleaved.Nucleophilic attack by the oxygen of the N-terminal neighboramide bond on the carbon center of the protonated amide bondleads to formation of a protonated oxazolone derivative (Yalcinet al., 1995, 1996; Nold et al., 1997; Paizs et al., 1999; Polce, Ren,& Wesdemiotis, 2000), whereas the detaching C-terminalfragment (amino acid or peptide) leaves the parent ion. Under

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low-energy collision conditions the loose complex of theprotonated oxazolone derivative and the leaving C-terminalfragment has a short but finite lifetime, and can undergo arearrangement which results in a proton–bound dimer of thesespecies (Paizs & Suhai, 2002a). Under such circumstances, theextra proton is shared by the two monomers, and dissociation ofthe proton-bound dimer will be determined by the thermo-chemistry (PA) of the species involved leading to ‘integrated’formation of bx and yz ions (Paizs & Suhai, 2002a). It is worthnoting here that the neutral counterparts of the yz ions on thebx� yz pathways are oxazolone derivatives. The dissociationkinetics of the dimer depends on the internal energy distributionof the ion population, the PA of its monomers, etc. and can beapproximated by using a linear free-energy relationship (Harri-son, 1999; Paizs & Suhai, 2002b, 2004):

lnbx

yz

� �� PAN-term � PAC-term

RTeff

� �ð1Þ

where bx/yz is the ratio of the abundances of the bx and yz ions,PAN-term and PAC-term are the proton affinities (PA) of the neutralfragments of the corresponding bx� yz pathway (i.e., PAs of anoxazolone derivative and a truncated peptide for the N- andC-terminal fragments, respectively), and Teff denotes the‘effective’ temperature.

Most of the experimental results obtained from structuraland energetic studies on small protonated peptides can beexplained based on mechanistic considerations involved in thebx� yz pathway. Harrison and co-workers (Yalcin et al., 1995)have investigated the CID spectra of protonated C6H5CO–Gly–Gly-OH. They found that the CID spectra of the b-type iongenerated from protonated C6H5CO–Gly–Gly-OH matches theCID spectra of protonated 2-phenyl-5-oxazolone supporting theoxazolone structure of the stable b ions.

yz ions have been found by tandem MS to be protonatedtruncated peptides or amino acids. The yz ions contain the addedproton as well as a hydrogen that was originally attached to a

SCHEME 3. (Continued )


515

nitrogen N-terminal to the cleaved amide bond and migrated tothe newly formed yz ion (Mueller, Eckersley, & Richter, 1988;Cordero, Houser, & Wesdemiotis, 1993). This proton migrationstep corresponds to the proton transfer reaction between theoxazolone derivative and the truncated peptide in the proton-bound dimer (Scheme 3a) on the bx� yz pathway. This protontransfer step was evaluated (Paizs, Suhai, & Harrison, 2003) forthe peptide H-Gly–Sar–Sar-OH (Scheme 3b) for which y1 ionscan not form on the b2� y1 pathway since the correspondingoxazolone derivative is a fixed charge and does not have a protonto be shared with the leaving H-Sar-OH. The breakdown graph ofH-Gly–Sar–Sar-OH is dominated by the b2 ion and y1 appearsonly at relatively high energies and originates very probably fromsecondary fragmentation of the y2 ion.

The N-terminus has also been probed by using neutralfragment reionization (NfR) (Nold et al., 1997) experiments,which give ambiguous results for the neutrals involved. Forprotonated C6H5CO–Gly–Phe-OH, the neutral counterpart ofthe y1 ion (protonated H-Phe-OH) is 2-phenyl-5-oxazolonesupporting the bx� yz pathway. On the other hand, NfR studies onprotonated H-Leu–Gly–Pro-OH have shown that the neutralcounterpart of the y1 ion is a diketopiperazine derivative, makingthe general applicability of the bx� yz pathway questionable. Wespeculate that the unusual behavior of H-Leu–Gly–Pro-OH isbecause of the proline effect and other tripeptides (having no Proat theC-terminal) fragment on the b2� y1 pathway. This questionis currently under investigation in our laboratory using bothexperimental and theoretical tools.

Morgan & Bursey (1994) have reported a linear relationshipbetween the logarithm of the ratio of y1 and b2 ion abundances(log(y1/b2)) and the PA of the C-terminal amino acid residue forprotonated tripeptides of the series H-Gly–Gly-Xxx-OH whereXxx was varied. (Fragmentation of all the H-Gly–Gly-Xxx-OHtripeptides leads to the same b2 ion, namely, protonated 2-aminomethyl-5-oxazolone.) The linearity of the log(y1/b2)versus PA of Xxx curve can be explained by considering thecorresponding b2� y1 pathway. As mentioned above, the finalstep of the bx� yz pathway corresponds to the dissociation of theproton-bound dimer of an oxazolone derivative and a truncatedpeptide. The dissociation kinetics can be approximated by usingEquation 1 in which PAN-term is constant (PA of 2-aminomethyl-5-oxazolone) whereas PAC-term is varied in the present caseexplaining the linearity of the ln(b2/y1) versus PA of Xxxrelationship observed experimentally. Furthermore, the combi-nation of relative ion abundances obtained from the low-energyMS/MS experiments for the H-Gly–Gly-Xxx-OH series oftripeptides with appropriate thermochemical data (protonaffinities of Xxx) gives (Paizs & Suhai, 2002b) a reasonable PAvalue for 2-aminomethyl-5-oxazolone. (This approach to deriv-ing thermochemical data is similar to Cooks’ kinetic method(McLuckey, Cameron, & Cooks, 1981).) Similar results havebeen obtained for other peptide series like H-Gly-Xxx–Phe-OH(Harrison, 2002), benzoyl-Gly–Gly-Xxx-OH (Morgan & Bur-sey, 1995), benzoyl-Xxx-Gly–Gly-OH (Morgan & Bursey,1995) providing reasonable PA values for intermediates (oxazo-lone derivatives, F, GG) occurring on various bx� yz pathways.This is possible only if charge-directed cleavage of theprotonated amide bonds of the investigated tripeptides isdominated by the bx� yz pathway.

General energetic, kinetic, and entropy factors determiningthe activity of the bx� yz pathways have recently beeninvestigated (Paizs & Suhai, 2004). The reactive configurationsof the bx� yz pathways (all-trans-amide nitrogen protonatedspecies) are energetically accessible under low-energy collisionconditions since the corresponding relative energies—calculatedwith respect to the most stable structure of the peptide protonatedat the most favored protonation site—are in the range of 15–25kcal/mol for peptides lacking arginine (Paizs et al., 1999, 2001;Csonka et al., 2001; Paizs & Suhai, 2001a,b; Jegorov et al., 2003).Theoretical studies (Csonka et al., 2000, 2001; Paizs et al., 2001;Paizs & Suhai, 2001b) on a few protonated peptides proved theexistence and facility of proton transfer pathways that connect themost favored and the amide nitrogen protonation sites at internalenergies well below the threshold energies of the lowestfragmentation pathways. Once the amide nitrogen protonatedspecies are reached, concerted formation of the oxazolone ringand cleavage of the amide bond takes place via a 10–15 kcal/molbarrier (Paizs et al., 1999; Paizs & Suhai, 2002a) on a time-scalecharacteristic of a rearrangement-type reaction. For the peptidesinvestigated so far, the energy level of the separated final productsis higher than the highest energy transition structure of thecorresponding bx� yz pathway. This finding is in line with theresults of metastable ion studies, which indicate small kineticenergy release (KER) values for the formation of bx and yz ionsof protonated peptides (Polce, Ren, & Wesdemiotis, 2000). Inmost of the amide nitrogen protonated species, the oxygen of theN-terminal neighbor amide bond takes part in charge solvation(CS) of the—NH2

þ—moiety bringing the nucleophilic oxygenclose to the positive carbon center. This ensures that entropyfactors do not preclude amide bond dissociation on the bx� yz

pathways.The mechanistic, energetic, and kinetic considerations

described above have been worked out for the fragmentationpathways of protonated pentaalanine (Paizs & Suhai, 2004)permitting for the first time a semi-quantitative understanding ofthe IIRs of the MS/MS spectra of a protonated oligopeptide. Theenergetics and kinetics of thevariousbx� yz pathways of protonatedpentaalanine have been determined by using theoretical methodswhich indicate that at low internal energies, the b4� y1 pathway isfavored compared to b3� y2 and b2� y3. This is in agreement withthe metastable ion and low-energy collision-induced dissociationmass spectra (Yalcin et al., 1996). At higher energies all the b4� y1,b3� y2, and b2� y3 PFPs are active behind secondary reactions likebn! bn� 1 and yn! yn� 1 (for details see below). Equation 1 wasused to approximate the ratio of the bx and yz ions on the particularbx� yz pathways. Applying the necessary proton affinities, suchconsiderations satisfactorily explain the dominance of the b4 ionover y1 and why the b3 ion is more abundant than y2 (both b3 and y2

are present in the mass spectra).

2. Diketopiperazine Peptide Fragmentation Pathways

All diketopiperazine pathways are initiated by mobilization ofthe added proton to form amide nitrogen protonated species.However, the mechanisms of the cleavages of the aa(2)–aa(3) andaa(n)–aa(nþ 1) (n> 2) amide bonds differ significantly, there-fore, these fragmentation pathways will be described separately.In general, we refer to the various ‘diketopiperazine’ pathways as

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diketopiperazine-yN� n, where N is the number of amino acidresidues in the peptide and n is used to note the amide bond(aa(n)–aa(nþ 1)) being cleaved.

The diketopiperazine-yN� 2 pathway (Scheme 4) leads tothe formation of diketopiperazine derivatives (Cordero, Houser,& Wesdemiotis, 1993) as the neutral counterparts of the yN� 2

ions. Since diketopiperazine derivatives contain two cis amidebonds, trans–cis isomerization of the initially trans N-terminal

amide bond (aa(1)–aa(2)) is necessary on the diketopiperazine-yN� 2 pathway (Paizs & Suhai, 2001b). The trans–cis isomer-ization involves species protonated at the nitrogen of the N-terminal amide bond (aa(1)–aa(2)) (Paizs & Suhai, 2001b). Thenext steps involve mobilization of the added proton to thenitrogen of the aa(2)–aa(3) amide bond (Scheme 4) and attack ofthe terminal amino group on the carbon center of the protonatedamide bond. A loose complex of the protonated diketopiperazine

SCHEME 4.


517

derivative and the leaving truncated peptide is then formed inwhich spontaneous proton transfer to the C-terminal fragmentoccurs because the PA of cyclic peptides is much smaller than thatof linear peptides (Nold, Cerda, & Wesdemiotis, 1999). Finally,the complex dissociates to form the y ion and a neutraldiketopiperazine derivative.

There is no need for cis–trans isomerization of amide bondson the diketopiperazine-yN� n (n> 2) pathways (Scheme 5) sincecyclic peptides, formed as the neutral counterparts of the yN� n

(n> 2) ions, can accommodate all of their amide bonds in thetrans isomerization state (Polce, Ren, & Wesdemiotis, 2000;Paizs & Suhai, 2004). Therefore, the diketopiperazine-yN� n

(n> 2) pathways are initiated by mobilization of the addedproton which is then followed by nucleophilic attack of the N-

terminal amino group on the carbon center of the protonatedamide bond. Formation of the cyclic peptide and cleavage of theamide bond take place in a concerted manner and yield primarilythe complex of the protonated cyclic peptide and the C-terminalfragment. Since the proton affinities of cyclic peptides are muchlower that that of linear peptides, the extra proton transfers to theC-terminal fragment and the complex dissociates to form theyN� n (n> 2) ion and the cyclic peptide as its neutral counterpart.The size of the cyclic peptide depends on how far the cleavedamide bond locates from the N-terminus. For yN� 3, yN� 4, andyN� 5 ions the corresponding neutrals are cyclo-tri-, cylo-tetra,and cyclo-penta-peptides, respectively.

Many of the experimental results obtained from structuraland energetic studies on small protonated peptides can be

SCHEME 5.

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518

explained based on mechanistic considerations involved in thediketopiperazine-yx pathways. yx ions have been found bytandem MS to be protonated truncated peptides or amino acids.The yx ions contain the added proton as well as a hydrogen thatwas originally attached to a nitrogen N-terminal to the cleavedamide bond and migrated to the newly formed yx ion (Mueller,Eckersley, & Richter, 1988; Cordero, Houser, & Wesdemiotis,1993). This proton migration step corresponds to the protontransfer reaction between the cyclic peptide and the truncatedpeptide in the complex on the diketopiperazine-yx pathways(Schemes 4 and 5). TheN-terminus has also been probed by usingneutral fragment reionization (NfR) (Nold et al., 1997) experi-ments which have shown that for protonated H-Leu–Gly–Pro-OH, the neutral counterpart of the y1 ion is a diketopiperazinederivative.

On the other hand, it seems to be rather difficult to explainthe linear relationship, found between the logarithm of the ratioof y1 and b2 ion abundances (log(y1/b2)) and the PA of the C-terminal amino acid residue for protonated tripeptides of theseries H-Gly–Gly-Xxx-OH, where Xxx was varied, consideringthe diketopiperazine-yN� 2 pathway. Wesdemiotis and co-work-ers (Nold, Cerda, & Wesdemiotis, 1999) have suggested thatprotonated oxazolones and cyclic peptides (like diketopiperazinederivatives) can interconvert in the ion/molecule complex formedby cleavage of the protonated amide bond in an energetically andkinetically accessible way (without significant barriers). There-fore, dissociation of the complex will be determined by theenergetics of the b2 and y1 exit channels (PA of the oxazolonesand C-terminal amino acids) explaining the linearity of thelog(y1/b2) versus PA of the Xxx for the series H-Gly–Gly-Xxx-OH. Paizs & Suhai (2001b) have shown, however, that theoxazolones and diketopiperazines formed from H-Gly–Gly-Xxx-OH differ significantly since the isomerization state of theN-terminal amide bond is cis and trans for these species,respectively. Since cis–trans isomerization of the amide bond istime-consuming and requires significant internal energy, it is,therefore, not likely that the protonated oxazolones and cyclicpeptides can interconvert in the ion/molecule complex formed bycleavage of the amide bond.

The energetics and the kinetics of the cleavage of the C-terminal amide bond of protonated tripeptides have beeninvestigated in detail comparing the diketopiperazine-yN� 2

and b2� y1 pathways (Paizs & Suhai, 2002a; Paizs, Suhai, &Harrison, 2003). For protonated H-Gly–Gly–Gly-OH, H-Gly–Gly–Sar-OH, and H-Gly–Sar–Sar-OH, the reactive configura-tions and the transition structures of the b2� y1 pathway (13–24and 23–29 kcal/mol relative energies, respectively) are morefavored than the corresponding diketopiperazine-yN� 2 values(23–30 and 30–37 kcal/mol relative energies, respectively). Onthe other hand, the energy level of the separated final products isalways much deeper for the diketopiperazine-y1 (18–26 kcal/mol relative energy) than for the b2� y1 (38–40 kcal/mol relativeenergy) pathway. If all the pre-dissociation and dissociationprocesses of the diketopiperazine-y1 and b2� y1 pathwaysoccurred under thermodynamic control and the kinetics did notprevent the formation of intermediates and the dissociation of theprotonated amide bonds, then the CID spectra of protonated H-Gly–Gly–Gly-OH, H-Gly–Gly–Sar-OH, and H-Gly–Sar–Sar-OH would be dominated by the y1 ion formed on the

diketopiperazine-y1 pathway. While the y1 ion dominates theCID spectrum of protonated H-Gly–Gly–Sar-OH, fragmenta-tion of protonated H-Gly–Sar–Sar-OH mainly leads to forma-tion of the b2 ion and the y1 ion appears only at higher energiesin the spectrum. This fact can be explained only by assuming thatthere is a step on the diketopiperazine-y1 pathway, which is underkinetic control. In our opinion, this rate-limiting step correspondsto a trans–cis isomerization of the N-terminal amide bond (Paizs& Suhai, 2001b).

Recent studies (Paizs & Suhai, 2004) on protonatedoligopeptides indicate the diketopiperazine-yx pathways arecontrolled by either energetic or kinetic or entropy factors in themajority of the cases. The diketopiperazine-yN� 2 pathways arekinetically controlled because trans–cis isomerization of the N-terminal amide bond has to take place prior to the nucleophilicattack (Paizs & Suhai, 2001b). In the case of the diketopiper-azine-yN� n (n is ‘average’) pathways trans–cis isomerization ofthe N-terminal amide bond is not necessary. The nitrogen of theterminal amino group can get close to the carbon center of theprotonated amide bond to initiate formation of the cyclic peptide.However, small cyclic peptides accommodating only trans amidebonds suffer from significant ring strain leading to energeticallydisfavored fragmentation products. As the size of the cyclicpeptide increases (cleavage far from the N-terminus), the cyclicpeptides will suffer from less and less ring strain leading toenergetically more favored diketopiperazine-yN� n (n is ‘large’)pathways. However, these diketopiperazine-yN� n pathways willbe discriminated by entropy effects. This is because of the factthat the amide nitrogen protonated species are effectivelysolvated by nearby amide oxygens and the terminal amino groupmust compete with this kind of CS to get close to the center of theprotonated amide bond. While CS of the—NH2

þ—moiety by theterminal amino group is energetically feasible, the number ofsuch species will be small compared to the large number ofsuch species where amide oxygens provide stabilization. Thismeans that dissociation of protonated oligopeptides on thediketopiperazine-yN� n pathways if the amide bond to be cleavedis located far from the N-terminus is disfavored because ofentropy factors.

3. Amide Oxygen Peptide Fragmentation Pathways

In the previous sections, it was repeatedly shown that amidenitrogen protonated species are responsible for cleavage of theamide bonds. This is because of the fact that protonation at thenitrogen weakens the amide bond and makes the carbon center ofthe amide bond more positive and therefore a possible target ofnucleophilic attack. Pathways like bx� yz and diketopiperazine-yx involve amide nitrogen protonated species as reactiveconfigurations. Modeling the energetic and kinetic details ofthe bx� yz pathways of protonated pentaalanine led to a semi-quantitative understanding of the MS/MS spectra of oligopep-tides. Contrary to the success of mechanisms that involve amidenitrogen protonated species, other pathways based on protona-tion at amide oxygens regularly appear in the literature. In thefollowing, we shortly describe the ‘‘amide oxygen’’ pathwaysand summarize why it is unlikely that these dissociation channelsare active under low-energy collision conditions.


519

It is well known that protonation at amide oxygens isenergetically favored compared with protonation at amidenitrogens. Based on this fact a mechanism (Arnot et al., 1994;Reid, Simpson, & O’Hair, 1998; Vaisar & Urban, 1998; Wysockiet al., 2000) was proposed for the formation of b and y ions whichdoes not involve any amide nitrogen protonated species. Themechanism applied to a general peptide is briefly outlined inScheme 6. The amide oxygen pathways are initiated bymobilization of the added proton to reach amide oxygens. Thisstep requires less energy than mobilization of the added proton toamide nitrogens. In the next steps, a five-membered ring isformed by attack of theN-terminal neighbor amide oxygen on thecarbon center of the carbonyl-O-protonated amide group, andthen an 1,1-elimination occurs resulting in loss of the C-terminalfragment and formation of a protonated oxazolone. TheN- andC-terminal fragments can separate with or without proton transferleading to b and y ions.

Theoretical investigation of the PES of a large number ofprotonated peptides (H-Gly–Gly–Gly-OH, H-Gly–Gly–Pro-OH, H-Ala–Gly–Gly-OH, H-Gly–Ala–Gly-OH, H-Gly–Gly–Ala-OH, H-Gly–Gly–Gly–Gly-OH, H-Ala–Ala–Ala–Ala–Ala-OH, etc.) indicates that amide oxygen protonated speciescontaining the five-membered ring exist but are energetically notfavored (Paizs & Suhai, 2001b). Actually, the relative energy ofsuch species is often higher than even those of the most stableamide nitrogen protonated species of the same compounds. Thenext step on the amide oxygen pathway corresponds to cleavageof the amide bond by 1,1 elimination on the Camide carbon of thealready high-energy amide oxygen protonated five-memberedring-containing species (Scheme 6). The corresponding transi-tion structure involves a highly strained four-membered ringleading to a large barrier (Paizs et al., 2001). For larger peptidesthis barrier could be reduced by catalysis of the proton transferstep involving other basic sites of the peptide. It seems likely,

SCHEME 6.

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however, that even after reducing the barrier, the correspondingtransition structures will be less favored than TSs on the bx� yz

pathways. These observations indicate that the amide oxygenpathways do not contribute significantly to the formation ofsequence ions of protonated peptides.

B. Dissociation of the N-terminal (aa(1)–aa(2)) AmideBond of Underivatized Protonated Peptides

Charge-directed dissociation of the underivatized N-terminalamide bond of protonated peptides significantly differs from thecleavage at other amide bonds of the peptide backbone.According to the PFP hierarchy of Scheme 2 described above,protonated amide bonds can be cleaved either by direct bondcleavage or by rearrangement-type reactions. Most of thefragmentation reactions of protonated peptides excited underlow-energy collision conditions belong to the latter class,because even the excited ions do not have enough energy tofragment on direct bond cleavage pathways which are facile onlyif significant energy is deposited into the ions. It is not surprisingthat low-energy pathways which include direct bond cleavage ofthe protonated amide bond are active mainly for the N-terminalamide bond (a1� yx pathway, Paizs & Suhai, 2001a) for whichthe only available backbone nucleophile that can inducefragmentation is theN-terminal amino group. The correspondingrearrangement-type reaction (aziridinone pathway, Cordero,Houser, & Wesdemiotis, 1993) however, includes the formationof an energetically disfavored three-membered ring giving rise tothe possibility of the a1� yx pathway to compete efficiently formainly dipeptides and small peptides containing amino acidresidues with high proton affinities at the N-terminus. In ouropinion, the majority of ions originating from dissociation of theN-terminal (aa(1)–aa(2)) amide bond of underivatized proto-nated peptides are formed on the a1� yx pathways. It is worthnoting here that one can easily shut down the a1� yx andaziridinone pathways by, for example, introducing throughacetylation of theN-terminus a good nucleophile (amide oxygen)N-terminal to the aa(1)–aa(2) amide bond that can already initiaterearrangement-type cleavage of the amide bond.

1. a1� yx Peptide Fragmentation Pathways

The major steps of the a1� yx pathway are illustrated inScheme 7. Being a charge-directed dissociation channel, thea1� yx pathway is initiated by mobilization of the added proton,which has to reach the nitrogen of the N-terminal amide bond.Getting through the a1� yx TS results in a trimer of a protonatedimine (N-terminal fragment), truncated peptide, or amino acid(C-terminal fragment) and CO. After loss of the weakly bondedCO, a proton-bound dimer of the N- and C-terminal fragments isformed. Under low-energy conditions the lifetime of this dimer islong enough so that numerous proton transfers can take placebetween the two fragments in the dimer. Therefore, there are twoexit channels through which the proton-bound dimer can dis-sociate without passing a barrier in the next step to form either a1

or yx ions. The dissociation kinetics of the dimer depends on theinternal energy distribution of the ion population, the PA of itsmonomers, etc. and can be approximated by using a linear free-

energy relationship (Harrison, 1999; Paizs & Suhai, 2002b; Paizset al., 2004):

lna1

yx

� �� PAN-term � PAC-term

RTeff

� �ð2Þ

where a1/yx is the ratio of the abundances of the a1 and yx ions,PAN-term and PAC-term are the proton affinities (PA) of the neutralfragments of the corresponding a1� yx pathway (that is, PAs ofan imine and a truncated peptide for the N- and C-terminalfragments, respectively), and Teff denotes the ‘effective’temperature.

The major characteristics of the a1� yx pathway can be usedto explain most of the corresponding experimental resultsobtained for protonated dipeptides and small peptides. yx ionshave been found by tandem MS to be protonated truncatedpeptides or amino acids. The yx ions contain the added proton aswell as an H-atom that was originally attached to the N-terminalnitrogen and migrated to the newly formed yx ion (Mueller,Eckersley, & Richter, 1988; Cordero, Houser, & Wesdemiotis,1993). This latter step corresponds to the proton transfer reactionbetween the N- and C-terminal fragments in the proton-bounddimer (Scheme 7) on the a1� yx pathway. On the other hand,when the C-terminus is eliminated as a neutral fragment, neutralfragment reionization (NfR) experiments have shown it to becleaved as an intact amino acid (Cordero, Houser, & Wesdemio-tis, 1993). While the original spectrum interpretation by Cordero,Houser, & Wesdemiotis is directed towards the aziridinone PFP(see below), the NfR spectrum of protonated H-Ala–Ala-OH(Cordero, Houser, & Wesdemiotis, 1993) is dominated by ionscharacteristic to the MeCH=NH imine produced as the neutralcounterpart of the y1 ion on the a1� yx pathway. The uncertaintyregarding the interpretation of the NfR spectrum could no doubtbe resolved if the corresponding experiments be repeated for asystem where the y1 is the major fragmentation product.

Harrison et al. (2000) have investigated the major low-energy fragmentation pathways of many protonated dipeptidespaying special attention to the various structural factors affectingthe a1/y1 abundance ratio. They have found that for a series ofprotonated dipeptides H-Val-Xxx-OH, ln(a1/y1) is a linearfunction of the PA of the variable C-terminal amino acid.(Originally, Harrison et al. plotted log(y1/a1) instead of ln(a1/y1).The log(y1/a1)! ln(a1/y1) transformation does not affect thelinearity of the plot, only the slope is changed.) As mentionedabove, the final step in the a1� yx pathway corresponds to thedissociation of the proton-bound dimer of an imine and an aminoacid for dipeptides (HN=CHCH(CH3)2 and Xxx, the variedamino acid for the H-Val-Xxx-OH series of peptides, respec-tively). The dissociation kinetics can be approximated by usingEquation 2 in which PAN-term is constant whereas PAC-term isvaried in the present case explaining the linearity of the ln(a1/y1)versus PA of Xxx relationship observed experimentally. Furthe-rmore, the combination of relative ion abundances obtained frommetastable ion fragmentation for the H-Val-Xxx-OH series ofdipeptides with appropriate thermochemical data (protonaffinities of Xxx) gives (Paizs et al., 2004) a reasonable PA valuefor HN=CHCH(CH3)2 (imine derived from Val). (This approachto derive thermochemical data is similar to Cooks’ kineticmethod (McLuckey, Cameron, & Cooks, 1981).) This is possible


521

only if charge-directed cleavage of the amide bond of theinvestigated dipeptides is dominated by the a1� yx pathway.Harrison et al. (2000) have investigated the fragmentation of theH-Xxx-Phe-OH dipeptide series for which ln(a1/y1) gave poorcorrelation with the PA of H-Xxx-OH. We have recently shown(Paizs et al., 2004) that reasonable correlation can be obtained ifone considers the ln(a1/y1) versus PA of the imines derived fromXxx relationship for the H-Xxx-Phe-OH data, in accordance withthe general characteristics of the a1� yx pathway. It is worthnoting here that Equation 2 can be used only for the low-energy

fragmentation regime since the proton equilibration implicit inthe corresponding mechanism may not be established if the lifetime of the proton bound dimmer is too short.

The energetics of the fragmentation of protonated dipeptideshas been investigated by using energy-resolved CID-MS(Klassen & Kebarle, 1997) and SID-MS (Laskin, Denisov, &Futrell, 2000). Klassen & Kebarle (1997) have determined thekinetic shift corrected appearance energy of the a1 ion of pro-tonated H-Gly–Gly-OH to be 43.7 kcal/mol. Laskin, Denisov, &Futrell (2000) have reported critical energies for the H-Ala–Ala-

SCHEME 7.

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OH�Hþ!H-Ala–Ala-OH�Hþ–CO, H-Ala–Ala-OH�Hþ! a1,and H-Ala–Ala-OH�Hþ! y1 reactions at 44.3, 48.7, and47.5 kcal/mol, respectively. These data are in keeping withcomputational results on the a1� yx pathway (Paizs & Suhai,2001a) showing that loss of CO is energetically more favoredthan formation of a1 or y1 for protonated H-Gly–Gly-OH. On theother hand, the theoretical and experimental data for formation ofa1 and y1 ions cannot be directly compared since the reactionpattern used to integrate the kinetics (Laskin, Denisov, & Futrell,2000) of the various dissociation channels did not include thea1� yx pathway and relied on direct formation of a1 and y1 fromthe parent ion. This suggests that caution must be observed inderiving energetic parameters from energy resolved MS studies,if the underlying reaction mechanisms are not fully known.

Theoretical investigation of the dissociation of the amidebond of a few dipeptides has indicated (Paizs & Suhai, 2001a;Paizs et al., 2004) that the transition structure belonging toconcerted cleavage of the amide and the Ca-Camide (CHR1-COfor Scheme 7) bonds lies at 32–38 kcal/mol relative energy andincreases in the investigated H-Val–Phe-OH, H-Val–Ala-OH,H-Ser–Ala-OH, H-Thr–Phe-OH, and H-Gly–Gly-OH series.The corresponding proton-bound dimers and separated CO andthe fully separated final products (either a1, C-terminal aminoacid and CO or y1, imine derived from theN-terminal amino acidand CO) have relative energies at 9–15 and 35–45 kcal/mol,respectively. This energetics is in keeping with the results ofmetastable ion studies which indicate small KER values forthe formation of a1 ions of protonated di- and tripeptides(Ambihapathy et al., 1997) and the y1 ion of H-Gly–Gly-OH(Polce, Ren, & Wesdemiotis, 2000).

The main characteristics of the a1� yx pathway can be alsoused to explain the energy-dependence of the mass spectra ofprotonated dipeptides. For example, unimolecular decomposi-tion of protonated H-Gly–Gly-OH (van Dongen et al., 1996)leads to y1 (35%), a1 (2%), b2 (24%) ions and loss of CO (38%).The abundance of these peaks change to 72, 17, 5, and 6% underlow-energy CID, from which it is evident that loss of CO and theformation of y1 ions are favored with respect the a1 ion. However,at higher energies the a1 ion becomes clearly dominant over y1

(Klassen & Kebarle, 1997) and the peak corresponding to CO lossdisappears. At very low energies many fragmenting specieswhich had enough energy to get through the a1� yx transitionstructure do not fully dissociate from the proton-bound dimerphase (CO loss peak) since this step requires significant energy.The fragmenting proton-bound dimers, which have relatively lowinternal energies choose the energetically favored way leading toy1 formation. As the internal energy increases one sees more y1

and a1 ions and less abundant CO loss. Finally, at high energiesthere is no time for proton-equilibration in the proton-bounddimer, formation of a1 will be favored with respect to that of y1.

2. Aziridinone Peptide Fragmentation Pathways

The major steps in the aziridinone pathway are illustrated inScheme 8. Being a charge-directed dissociation channel, theaziridinone pathway is initiated by mobilization of the addedproton, which has to reach the nitrogen of the N-terminal amidebond. The aziridinone transition structure corresponds toconcerted cleavage of the protonated amide bond and formation

of the aziridinone ring. Under low-energy collision conditionsthe N- and C-terminal fragments (an aziridinone derivative and atruncated peptide, respectively) do not separate immediately andform a proton-bound dimer (Harrison et al., 2000) in whichnumerous proton transfers can occur between the two monomers.(The nitrogen protonated aziridinone derivative is stable in theproton-bound dimer (Harrison et al., 2000).) Dissociation of theproton-bound dimer leads to yx ions if the C-terminal fragmentkeeps the added proton during spatial separation of thefragments. On the other hand, the nitrogen protonated aziridi-none derivative is not stable (Harrison et al., 2000) and decom-poses to a1 and CO if the extra proton is kept by the N-terminalfragment. (The O-protonated form of aziridinone is stable but theN-protonated isomer is formed in the aziridinone pathway.)

Mechanistic considerations involved in the aziridinonepathway can explain some of the experimental results obtainedfor protonated dipeptides and small peptides. yx ions have beenfound by tandem MS to be protonated truncated peptides oramino acids which contain the added proton as well as an H-atomthat was originally attached to the N-terminal nitrogen andmigrated to the newly formed yx ion (Mueller, Eckersley, &Richter, 1988; Cordero, Houser, & Wesdemiotis, 1993). Migra-tion of the proton on the aziridinone pathway corresponds to theproton transfer reaction between the N- and C-terminal frag-ments in the proton-bound dimer of the aziridinone derivative andthe truncated peptide (Scheme 8) and involves one of thehydrogens originally attached to the terminal amino group. Theaziridinone pathway is in line also with the results of NfRexperiments which show that if the C-terminus is eliminated as aneutral fragment then it is cleaved as an intact amino acid or smallpeptide (Cordero, Houser, & Wesdemiotis, 1993). NfR experi-ments on protonated H-Ala–Ala-OH gave some support to theaziridinone pathway since the NfR spectra contained low-abundance peaks that are signature ions of the correspondingaziridinone derivative.

The aziridinone pathway could explain also the linearity ofthe log(a1/y1) versus PA of Xxx curves for the series of H-Val-Xxx-OH dipeptides (Harrison et al., 2000) by considering thatdissociation of the proton-bound dimer of the aziridinonederivative and the C-terminal fragment is determined by theproton affinities of the corresponding neutrals. Unfortunately,this hypothesis cannot be quantitatively assessed as protonaffinities are available neither experimentally nor computation-ally for aziridinone derivatives since the N-protonated form is notstable. On the other hand, combination of relative ion abundancesobtained from low-energy MS/MS experiments for the H-Val-Xxx-OH series of dipeptides with appropriate thermochemicaldata (proton affinities of Xxx) gives (Paizs et al., 2004) areasonable PAvalue for the intermediate (HN=CHCH(CH3)2) onthe corresponding a1� yx pathway. Since it is not likely that thePAs of HN=CHCH(CH3)2 and the corresponding aziridinonederivative in the proton-bound dimer of the aziridinone pathwayare equal, this fact strongly suggests that only a small fraction ofthe a1 and y1 ions are formed on the aziridinone pathway.

Theoretical investigation of the dissociation of the amidebond of a few dipeptides indicated (Paizs & Suhai, 2001a; Paizset al., 2004) that the aziridinone pathway is both energetically andkinetically less favored than the corresponding a1� yx pathways.The aziridinone transition structures lie at 44–50 kcal/mol


523

relative energies for H-Val–Phe-OH, H-Val–Ala-OH, H-Ser–Ala-OH, H-Thr–Phe-OH, and H-Gly–Gly-OH. RRKM calcula-tions suggest that the corresponding unimolecular rate constantsare much smaller than the corresponding a1� yx values. Further-more, the separated final products are energetically disfavored onthe aziridinone pathway compared to a1� yx if yx ions areformed. (The three-membered aziridinone ring is energeticallymore demanding than separated CO and the correspondingimine.)

There seems to be a controversy between the mechanisticconsiderations involved in the aziridinone pathway and thetendency to form [MH�CO]þ ions for some protonateddipeptides. Formation of a1 ions on the aziridinone pathway isproposed via separation of the proton-bound dimer to form thenitrogen protonated aziridinone derivative, which is stable onlyin the complex and decomposes as the C-terminal fragmentrepels out. It is not likely that [MH�CO]þ ions are formed afterthe dissociation of the protonated aziridinone by reunion of theprotonated imine (a1) and theC-terminal fragment. Also, it seemsto be rather difficult to explain the energy dependence of the MS/

MS spectra of dipeptides like H-Gly–Gly-OH based on theaziridinone pathway.

C. Charge Directed Amide Bond Cleavage via Attackof Nucleophilic Groups of Amino Acid Side Chains

Beside the PFPs involving interaction of only backbonefunctional groups, some peptides can dissociate via amide bondcleavage initiated by specific amino acid side chains (Scheme 9).The most important such reactions involve nucleophilic attack bythe histidine, glutamine, asparagine, lysine, and arginine sidechains on the C-terminal adjacent nitrogen protonated amidebond and lead to various cyclic (non-oxazolone derivative) C-terminal fragments. Since the reactive configurations of thecorresponding side chain initiated and bx� yz pathways(Scheme 9) are species containing the nitrogen protonated amidebond, competition of the two dissociation channels can lead tomixture of the two isomeric forms of the N-terminal fragments.While the charge directed dissociation of amide bonds is

SCHEME 8.

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dominated in most of the cases by the general bx� yz pathways,side chain induced cleavage C-terminal to histidine andglutamine can also be significant in some cases.

1. Histidine Effect

The PFP behind the histidine effect is a charge-directed pathway,which involves active involvement of the His side chain insteadof backbone nucleophiles like the amide oxygens or the nitrogenof the terminal amino group. The histidine effect has been studiedby Wysocki et al. (2000) and Farrugia, Taverner, & O’Hair (2001)in detail.

Cleavage at the C-terminal side of histidine is preferred formany peptides if the number of added protons is larger than thenumber of Arg residues present (Wysocki et al., 2000). Becauseof its high PA the His side chain is very probably protonated in thelowest energy structures of such peptides (Scheme 10). The firststep of the reaction mechanism involves mobilization of theproton located at the His side chain to the nitrogen of the C-terminal neighbor amide bond, which is followed by nucleophilicattack of the imidazole nitrogen on the carbon of the protonatedamide bond. The bx ion formed in this reaction is a bicyclic ion(Scheme 10) and does not have the classical oxazolone structure.Under low-energy collision conditions the life time of the

complex of the N- and C-terminal fragments is long enough toallow proton transfer between the monomers and formation ofboth bx and yz ions.

The histidine effect has been systematically probed(Wysocki et al., 2000) by MSn experiments, varying the adjacentamino acid residues in the peptide, alkylation of the His sidechain, etc. For example, the MS/MS spectrum of doublyprotonated H-Arg–Val–Tyr–Ile–His–Pro–Phe-OH is domi-nated by the b5

þ and y2þ ion pair suggesting that the proton

transfer step of Scheme 10 is possible. Substituting Pro by Alaresults in dominance of b5

2þ and b62þ. This can be explained by the

difference between the PAs of H-Pro–Phe–OH and H-Ala–Phe-OH. Finally, alkylation of the His side chain shuts down theproton transfer part of the mechanism depicted in Scheme 10leading to dominance of bx

2þ ions in the MS/MS spectra. Also,ab initio calculations indicate that the non-classical bx ion ofScheme 10 is stable (Farrugia, Taverner, & O’Hair, 2001).

2. Peptide Fragmentation Pathways Involving theAmide Oxygen of the Gln and Asn Side Chains

The amide moieties of the Gln and Asn side chains can beinvolved in reactions leading to cleavage of the amide bond C-terminal to these amino acid residues. The corresponding PFPs

SCHEME 9.


525

are initiated by mobilizing the added proton which should reachthe nitrogen of the amide bond to be cleaved (Schemes 9 and 11).Nucleophilic attack by the amide oxygen of the Gln or Asn sidechain on the carbon center of the protonated amide bond leads toformation of cyclic isoimide (Jonsson et al., 2001a,b; Farrugia,O’Hair, & Reid, 2001; Harrison, 2003) derivatives, whereas thedetaching C-terminal fragment (amino acid or peptide) leavesthe parent ion. The same amide bond can also be cleaved on thecorresponding bx� yz pathway which leads to oxazoloneformation and therefore raises the possibility of forming isomersof the N-terminal fragment (Scheme 9). Under low-energycollision conditions the loose complex of the protonated isoimidederivative and the leaving C-terminal fragment has a short butfinite lifetime allowing rearrangement of and proton transfersbetween the fragments. Therefore, dissociation of the dimer will

be determined by the thermochemistry (PA) of the speciesinvolved leading to ‘integrated’ formation of bx and yz ionssimilarly to the bx� yz pathways.

Farrugia, O’Hair, & Reid (2001) have investigated b2 ionsderived from protonated N-acyl Gln and Asn methyl esters usingmultistage MS and ab initio techniques. These b2 ions fragmentby losing both CO (for more details, see the chapter on thebx! ax pathway) and CH2CO, indicating that both the oxazoloneand the isoimide forms are present in the mass spectrometer. Abinitio calculations have indicated that the oxazolone form is morestable than the isoimide isomer for both the Gln and Asncontaining b2 ions. However, the difference between the relativeenergies of the two forms is much smaller for the former(�2 kcal/mol) than for the latter (�11 kcal/mol). Cleavage of theamide bond C-terminal to Asn and Gln involving nucleophilic

SCHEME 10.

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attack of the side chain amide nitrogen has also been investigatedby determining the relative energies of the corresponding isomersof the b2 ions. These calculations have indicated that such areaction is energetically disfavored.

Harrison (2003) has investigated the fragmentation path-ways of protonated H-Gly–Gln–Gly-OH using multistage MS.The b2 ion shows a variety of fragmentation reactions includingloss of CO (for more details, see the chapter on the bx! ax

pathway) and a glycine residue. The former reaction ischaracteristic of b ions with the classical oxazolone structurewhereas the latter dissociation channel can be derived only from

the isoimide isomer. Jonsson et al. (2001a,b) have investigatedthe fragmentation reactions of tryptic and synthetic peptidescontaining the Gln–Gly sequence motif. These authors foundthat facile Gln–Gly cleavage occurs when an Xxx-Gln–Gly-Yyysequence is present in the peptide, where Xxx is any amino acidand Yyy is any amino acid except for Gly. Also, a model peptidecontaining Asn instead of Gln showed less dominant dissociationof the Xxx-Gly amide bond in line with the theoretical dataobtained by Farrugia, O’Hair, & Reid (2001) on the stability ofthe oxazolone and isoimide forms of the b2 ions derived from theN-acyl Asn and Gln methyl esters.

SCHEME 11.


527

3. Peptide Fragmentation Pathways Involvingthe Lys and Arg Side Chains

Peptides containing only Lys as a basic amino acid are mostprobably protonated at the e-amino group of the Lys side chain.Mobilization of the added proton can lead to various amidenitrogen protonated species resulting in formation of bx and yz

ions on the bx� yz pathways. If the extra proton reaches the Lys-Xxx amide bond, the side chain of Lys can also initiate amidebond cleavage to form a caprolactam derivative (Scheme 12).Yalcin & Harrison (1996) have investigated the fragmentationpathways of protonated H-Lys-Gly-OH and other Lys deriva-tives. The nominal acylium ion at m/z¼ 129 is produced in bothmetastable ion and collision induced fragmentation of theinvestigated Lys derivatives. The product ion spectra of ionm/z¼ 129 is very similar to that of protonated a-amino-e-caprolactam confirming the dissociation mechanism of Scheme12. The PFPs of protonated H-Lys-Gly-OH have also beenstudied by using quantum chemical and RRKM calculations(Csonka et al., 2001) further confirming the assumption ofcaprolactam formation.

Farrugia, O’Hair, & Reid (2001) have investigated the b2 ionderived from protonated N-acyl Lys methyl ester using multi-stage MS and ab initio techniques. The corresponding b2 ion does

not lose CO (for more details, see the chapter on the bx! ax

pathway) at all, indicating that only the caprolactam form ispresent in the mass spectrometer. Ab initio calculations havesuggested that the oxazolone form (Scheme 9) is less stable thanthe caprolactam isomer by �2 kcal/mol.

Kish & Wesdemiotis (2003) have investigated the PFPs ofprotonated H-Gly–Gly–Lys–Ala–Ala-OH utilizing multistageMS in an ion trap instrument. Formation of the corresponding b3

ion is more significant than that of the b3 ion of protonated H-Tyr–Gly–Gly–Phe–Leu-OH. The b3 ion derived from H-Gly–Gly–Lys–Ala–Ala-OH fragments by losing H2O and Gly–Glywith no signs of dissociations (loss of CO and the bx! bx� 1

channel, see later) characteristic to the oxazolone structure.These findings can be rationalized again by considering thecaprolactam (Scheme 13) structure of the b3 ion.

The b2 ion derived from protonated N-acyl Arg methyl esterhas recently been investigated using multistage MS and ab initiotechniques (Farrugia, O’Hair, & Reid, 2001). The MS/MS/MSspectrum does not show CO loss indicating a non-classicalstructure of the b2 ion. This behavior is in line with themechanism depicted in Scheme 13 which involves mobilizationof the extra proton originally sequestered by the Arg side chain tothe Arg-Xxx amide nitrogen and subsequent nuclephilic attackby the neutral guanidino group to induce amide bond cleavage

SCHEME 12.

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and formation of a six-membered ring. Ab initio calculations(Farrugia, O’Hair, & Reid, 2001) have suggested that theoxazolone form (Scheme 9) is less stable than the non-classical b2 isomer by � 2 kcal/mol. The MS/MS spectrum ofprotonated H-Arg–Asp-NH2 shows an abundant peak atm/z¼ 157 (Paizs et al., 2002) representing the usually unstableb1 ion, whose formation can be explained by the mechanism ofScheme 13.

It is worth noting here that Farrugia & O’Hair (2002) haverecently discovered an interesting gas phase rearrangement forprotonated arginine-containing dipeptides. For protonated H-Arg–Gly-OH and H-Gly–Arg-OH, the rearrangement leads toidentical MS/MS spectra. DFT calculations and MS/MS/MSexperiments suggest a mechanism, which involves formation of acommon cyclic intermediate. Recent MS/MS results on proto-nated H-Arg–Gly–Gly-OH and H-Gly–Gly–Arg-OH indicatethat this rearrangement is characteristic of dipeptides, and thatlarger Arg-containing peptides fragment by involving other PFPs(Paizs & Somogyi, unpublished results).

D. Peptide Fragmentation Pathways Leadingto Loss of Small Neutrals

The MS/MS spectra of protonated peptides often containsfragment ions originating from losses of small neutrals likewater and ammonia from the parent and various fragment ions.Under low-energy conditions the [MH�H2O]þ, [MH�NH3]þ,[yz�H2O]þ, [yz�NH3]þ, [bx�H2O]þ, [bx�NH3]þ ions areformed on charge-directed PFPs. There are three possibilities forthe water loss (Ballard & Gaskell, 1993) of protonated peptidesinvolving dehydration of the C-terminal, Asp, and Glu COOHgroup, at backbone amide oxygens, and at side chains of Ser andThr. For some peptides only one of the corresponding pathwaysdominates whereas the fragmentation of other peptides showmixing of the above channels. Loss of ammonia occurs from theside chains of Arg, Lys, Asn, and Gln. These reactions arepractically important since tryptic peptides contain either Arg orLys at the C-terminus leading to usually abundant yz-NH3 series.It is worth noting here that elimination of CO can formally be

SCHEME 13.


529

considered as a PFP leading to loss of a small neutral. There aretwo practically important cases involving loss of CO from bx ionsand from intact peptide ions. The former reaction is discussed in‘‘bx! ax Pathways.’’ whereas the latter is described in ‘‘a1� yz

Pathways.’’

1. Water-Loss Peptide Fragmentation Pathways

Loss of water from the C-terminal COOH group is initiated bymobilization of the extra proton, which has to reach the hydroxylgroup. According to the theoretical study by Balta, Aviyente, &Lifshitz (2003) this proton transfer pathway involves relatively

low-energy amide oxygen protonation sites (Scheme 14, route 1).Once the added proton reached theC-terminus, proton transfer tothe OH group, breaking of the HO–C bond, and formation of anoxazolone ring occur concertedly but asynchronously. The firsttwo events occur at the early stage of the fragmentation whereasoxazolone formation takes place only at the final stage of thereaction. Multistage MS and H/D exchange experiments by Reid,Simpson, & O’Hair (1999) proved the expected oxazolonestructure of the b2 and b3 ions derived from protonated H-Gly–Gly-OH and H-Gly–Gly–Gly-OH. Ballard & Gaskell (1993)have investigated the water loss PFPs of various peptides utilizing[18O] labeling and/or blocking of the C-terminal carboxyl group.

SCHEME 14.

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These investigations indicated that protonated H-Thr–Arg–Lys–Arg-OH dehydrates nearly exclusively at the C-terminalCOOH group. Water loss from the Asp and Glu side chains occursvia a similar mechanism shown in Scheme 14 (route 1) and themain difference between the reactions of the backbone and sidechain COOH groups is in the structure of the resulting cyclicproducts. A practically significant side chain activity occurs ifpeptides and/or their yz ions contain Glu at the N-terminalposition. For such cases, formation of pyroglutamic acid(Scheme 14, route 2) is usually facile and accompanied byabundant loss of water.

The MS/MS spectra of protonated [18O2] H-Arg–Pro–Pro–Gly–Phe-OH (Ballard & Gaskell, 1993) show that dehydrationoccurs primarily through the loss of H2

16O involving one or moreof the backbone amide oxygens. MS/MS/MS studies indicatedthat the primary site of water loss involves the carbonyl oxygen ofthe amide bond nearest to the N-terminus. Reid, Simpson, &O’Hair (1999) have proved that the b4 and b5 ions derived fromprotonated H-Gly–Gly–Gly–Gly-OH and H-Gly–Gly–Gly–Gly–Gly-OH do not have oxazolone structure which is expectedif dehydration involves the C-terminal COOH group. Also, theMS/MS spectrum of methylated H-Gly–Gly–Gly–Gly-OHsuggests that elimination of water occurs involving an amideoxygen. While some efforts to elucidate the mechanism behindthe backbone water loss are described (Reid, Simpson, & O’Hair,1999), atomistic details of the underlying chemistry are notknown yet.

Dehydration involving the Ser and Thr side chains has beenstudied by Ballard & Gaskell (1993) and Reid, Simpson, &

O’Hair (2000). The corresponding reaction (Scheme 15) isinitiated by mobilization of the extra proton to the Ser or Thr sidechain oxygen which is followed by nucleophilic attack of theC-terminal adjacent amide oxygen to form a stable five- or six-membered ring, respectively. Protonated tris-methyl esterderivative of the delta-sleep-inducing peptide fragments via lossof water despite its lack of free carboxylic acid groups (Ballard &Gaskell, 1993). Analysis of the MS/MS/MS spectrum andprotecting the Ser OH group suggest that water is eliminatedfrom the serine side chain for this peptide.

2. Ammonia-Loss Peptide Fragmentation Pathways

Charge directed loss of ammonia occurs from the side chains ofAsn, Gln, Lys, and Arg amino acid residues. (No data are reportedon ammonia loss involving the N-terminal amino group in theliterature.) A common characteristic of the ammonia loss PFPs isthat protonation at the corresponding side chain is required. Forthose cases when NH3 is eliminated from the Lys and Argside chains, mobilization of the extra proton is not required sincethe corresponding e-amino and guanidino groups are usually themost favored protonation sites of peptides. For deamidation fromthe Asn and Gln side chains, mobilization of the extra protonfrom more favored protonation sites like the N-terminal aminogroup of basic amino acid side chains is necessary.

Loss of ammonia occurs from Lys-containing peptides viaSN2-type reactions (Csonka et al., 2001) which lead toelimination of the nitrogen of the e-amino group. (15N-labelingexperiments (Dookeran, Yalcin, & Harrison, 1996) have shown

SCHEME 15.


531

that elimination of ammonia specifically involves the nitrogen ofthe side chain of protonated H-Lys-OH.) If Lys is located at theN-terminus of the peptide under investigation, the most likely PFP(Csonka et al., 2001) involves the nucleophilic attack by the N-terminal amino group (Scheme 16, reaction 1) leading toformation of pipecolic acid at the N-terminus. It is worth notinghere that nucleophilic attack by the Lys-Xxx amide oxygen toinduce NH3 loss from the Lys side chain is also possible but thispathway is kinetically disfavored with respect to the PFPinvolving the a-amino group.

If Lys is located far from the N-terminus, entropy factorspreclude elimination of ammonia via nucleophilic attack by theN-terminal amino group. In such circumstances, ammonia loss isinitiated by the Lys-Xxx amide oxygen or the C-terminal COOHgroup (Scheme 16, reaction 2). This PFP is of practicalimportance when tryptic peptides with Lys at the C-terminusare dissociated.

Elimination of NH3 from the protonated guanidino group ofthe Arg side chain has recently been investigated by usingtheoretical methods (Csonka, Paizs, & Suhai, 2004, Modeling ofthe gas-phase ion chemistry of protonated arginine J MassSpectrom, accepted for publication). These calculations indicatethat beside the usual NeH–Cþ(NhH2)(Nh0H2) form of the

protonated guanidino group other tautomers like –NeH–C(NhH3

þ)(Nh0H) or –Ne–C(NhH3þ)(Nh0H2) (Scheme 17) invol-

ving the preformed—NH3 group can exist in mass spectrom-eters. Loss of ammonia from the Arg side chain occurs veryprobably by SN1 substitution on the central z-carbon of theguanidine group from the –NeH–C(NhH3

þ)(Nh0H) or –Ne–C(NhH3

þ)(Nh0H2) tautomers. The resulting protonated carbodi-imide group (Scheme 17) can be stabilized by intramolecularattack of nearby nucleophiles leading to ring formation.Theoretical studies on protonated H-Arg-OH indicate thispathway requires at least �45 kcal/mol internal energy. It isworth noting here that BIRD of protonated bradykinin (Price,Schnier, & Williams, 1996) leads to elimination of NH3 with 1.3eVactivation energy. The theoretical value for protonated H-Arg-OH and the BIRD data for protonated bradykinin differsignificantly, suggesting that either SB structure of protonatedbradykinin (Schnier et al., 1996) influences the energetics of thePFP depicted in Scheme 17 or an alternative lower-energy routeexists for the elimination of NH3 from the Arg side chain.

Loss of ammonia from the Asn and Gln residues is initiatedby mobilization of the extra proton to the nitrogen of the sidechain amide bond, the carbon center of which becomes a likelytarget of nucleophilic attack. If Gln is located at the N-terminus,

SCHEME 16.

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532

the a-amino group can be involved as attacking nucleophileleading to facile pyroglutamate formation (Scheme 18, reaction1). If Gln and Asn are located far from the N-terminus, amideoxygens can initiate the deamidation (Scheme 18, reaction 2).The corresponding chemistry is complicated and involvesformation of rather unexpected products (Harrison, 2003). Forexample, elimination of ammonia from protonated H-Gly–Gln-Gly-OH and H-Gly–Gly–Gln-OH (Scheme 18, reaction 3) leadsat least partially to formation of protonated H-Gly–Gln–Pyr-OH

and H-Gly–Gly–Pyr-OH, respectively, in a reaction with a yetunknown mechanism.

E. Fragmentation Pathways of bx and yz Ions

The MS/MS spectra of protonated peptides frequently showwhole or partial series of bx and yz ions. As discussed above, theprimary source of these ions is the peptide parent ion. However,bx and yz ions can also be formed from higher b and y ions

SCHEME 17.


533

(Ballard & Gaskell, 1991; Yalcin et al., 1996). The yz ions aretruncated peptides, their fragmentation behavior can bedescribed according to the rules of the bx� yz, a1� yz, etc.,PFPs as already discussed. On the other hand, the majority of bx

ions contain an oxazolone ring at the C-terminus which leads tospecific fragmentation channels like elimination of CO (bx! ax

pathway) and formation of the next lower b ion (bx! bx� 1

pathway).It is well-known that the MS/MS spectra of the practically

important doubly protonated tryptic peptides show relativelyweak bx ion signals when acquired in triple quadrupole orquadrupole time-of-flight (Q-TOF) mass spectrometers. This

SCHEME 18.

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behavior is very probably because of facile bx! bx� 1 pathwayswhich tend to empty the higher and to populate the lower b ions.On the other hand, singly charged yz ions (co-produced with bx)of tryptic peptides seem to be more stable (no facile yz! yz� 1

transitions) because the added proton is sequestered by the C-terminal Lys or Arg side chains.

It is also worth noting here that formation of internalfragments and internal immonium ions is possible from both bx

and yz ions.

1. bx! bx� 1 Pathways

The bx! bx� 1 pathways have been studied in detail by theHarrison and Glish groups (Yalcin et al., 1996; Vachet, Ray, &Glish, 1998). Harrison and co-workers have investigated thereactions of higher bx ions of protonated H-Gly–Gly–Gly–Gly-OH, H-Gly–Gly–Gly–Gly–Gly-OH, and H-Tyr–Gly–Gly–Phe–Leu-OH. Depending on the amino acid composition, b3

and b4 ions fragment in part to form the next-lower b ions inreactions occurring with relatively low KERs on the metastableion time scale. Based on the observed fragmentation character-istics, it was proposed that the investigated bx ions have theprotonated oxazolone structure. This suggestion has recentlybeen confirmed in a theoretical study on the bx ions of protonatedpentaalanine (Paizs & Suhai, 2004).

However, the atomistic details of the underlying reactionmechanism are not yet clarified. Harrison suggested (Yalcin et al.,1996) a one-step mechanism in which the C-terminal adjacentamide oxygen attacks the –C=Nþ– carbon of the oxazolonering and induces elimination of an aziridinone derivative(Scheme 19). Fang et al. (1999) have studied this mechanismutilizing theoretical methods and determined the barrier to thecorresponding transition structure at �77 kcal/mol which isclearly too high for mass spectrometers operating under low-energy collision conditions. These authors have also investigatedthe bx! ax! bx� 1 indirect pathway (Scheme 19, route 2) whichrequires at least �40 kcal/mol internal energy and seems to bemore favored than the direct bx! bx� 1 pathway of Scheme 19(route 1). However, metastable ion studies by Yalcin et al. (1995)showed that the bx! ax reaction occurs with substantial releaseof kinetic energy (T1/2¼ 0.3–0.5 eV) indicating that the indirectbx! ax! bx� 1 pathway cannot be responsible for the formationof bx ions which is usually accompanied with low KER values.Also, Vachet, Ray, & Glish (1998) have studied the bx! bx� 1

pathways by using stored waveform inverse Fourier transformand double resonance techniques in conjunction with a quadru-pole ion trap. By ejecting some product ions as they are formed,further dissociation of these ions can be systematicallyinvestigated in this way. Vachet, Ray, & Glish (1998) showedthat �50 % of the b3 ions of various Leu-enkephaline derivativesoriginated directly from the corresponding b4 ions under themoderately energetic conditions applied. These experimentaldata clearly suggest that another (yet unknown) direct bx! bx� 1

pathway must exist.

2. bx! ax Pathways

The bx! ax pathways often produce bx and ax ion pairs separatedby the characteristic 28 Da mass difference which facilitates

identification of members of the b ion series in the MS/MSspectra of protonated peptides. Also, under low-energy collisionconditions the bx! ax pathway (Scheme 20) is the main source ofax (immonium) ions that could dissociate further to form internalfragments and/or internal immonium ions.

Protonation of the nitrogen of the oxazolone ring leads toweakening of the –O–CO– bond. Elimination of CO occurs on aconcerted pathway (Paizs et al., 2000) involving rupture of twocovalent bonds of the cyclic bx ion and leads to the open ax ion.The barrier height for the bx! ax pathways of HCO–NH–CH2COþ, MeCO–NH–CHMeCOþ, NH2–iBuCH–CO–NH–CH2–COþ, and NH2–CH2–CO–NH-i-BuCHCOþ b2

þ ions wasfound to be 29.0, 30.5, 34.6, and 30.3 kcal/mol, respectively. Thebx! ax TSs are energetically less favored than the separated finalproducts leading to 3.7, 14.6, 9.6, and 18.4 kcal/mol reverseactivation barriers for the b2 ions listed above (Paizs et al., 2000).

This energetics is in keeping with the experimental fact thatbx ions fragment on the metastable ion time scale by eliminationof CO with substantial KER (Yalcin et al., 1995, 1996),suggesting that the stable form of bx ions fragment through atransition structure that is higher in energy than the final products.It is worth noting here that El Aribi et al. (2003) have recentlyshown that the linear a2 ions can be further stabilized bynucleophilic attack of the N-terminal amino nitrogen on thecarbon of the immonium moiety, forming a five-memberedring. Also, some b2 ions tend to fragment to a1 ions (bx! ax� 1

pathway, Ambihapathy et al., 1997). However, the bx! ax� 1

pathway seems to be disfavored with respect to the bx! bx� 1

and bx! ax dissociation channels for larger b ions.

3. Formation of Internal Fragments

The formation of internal fragments requires cleavage of at leasttwo backbone amide bonds and leads to ions with underivatizedN- and oxazolone or immonium C-terminus. The majorcharacteristics of amide bond cleavage are described in detailin the previous sections during the discussion of the bx� yz,a1� yz, etc. PFPs, the same rules apply to the internal fragmentdissociation channels as well. It must be noted that internalfragments appear in the low-energy collision induced MS/MSspectra of protonated peptides in mostly those cases whenunusually abundant y ions are present. This situation occurs mostfrequently for peptides containing Pro and His (for more details,see sections on the proline and histidine effects). This finding is inline with the fragmentation behavior of higher bx ions, which tendto fragment on the bx! bx� 1 and bx! ax pathways instead ofcleavage of intact amide bonds.

Ballard & Gaskell (1991) have investigated formation ofinternal fragments of protonated H-Tyr–Gly–Gly–Phe–Leu-OH and des-Arg1-substance P using sequential product ionscanning and reaction intermediate scanning experiments. Bothprotonated H-Tyr–Gly–Gly–Phe–Leu-OH and des-Arg1-sub-stance P fragment, resulting in abundant internal fragments underthe mass spectrometric conditions applied. For both peptidescertain yz ions show high inherent tendencies to fragment furtherto give internal fragments. For example, internal fragments ofdes-Arg1-substance P belong to the axy8 and bxy8 classes formedby cleavage at the Lys–Pro amide bond (proline effect). Whilethe bx ions are also present in the MS/MS spectrum of protonated


535

des-Arg1-substance P, reaction intermediate scans indicate thatthe internal fragments are mainly originating from the y8 ion.

4. Pathways Leading to Internal Immonium Ions

Immonium ions of the N-terminal amino acid residue ofprotonated peptides can be formed on the a1� yz and b2! a1

pathways described above. However, the low-mass region of theMS/MS spectra often contains immonium ions originating fromamino acid residues located at other positions. Formation of suchinternal immonium ions requires cleavage of at least two amidebonds similarly to the dissociation channels leading to internalfragments. (Formally, internal immonium ions are (anyN� nþ1)1

type internal fragments.)

There are two major pathways, which lead to internalimmonium ions (Ix). Ambihapathy et al. (1997) have found thatax ions—formed on the bx� yz and bx! ax pathways as shown inScheme 21—tend to fragment further to ax� 1 and Ix ions on theax! ax� 1 pathway. In a recent study, El Aribi et al. (2003) haveshown that the ax! ax� 1 pathway can be considered as a suitablegeneralization of the a1� yz pathway (Paizs & Suhai, 2001a)since concerted cleavages of the Ca–CO and OC–N bonds occurin both cases in the –HCa–CO–NH2

þ– and –HCa–CO–NHþ=moieties, respectively. That is, after repelling out CO a proton-bound dimer of ax� 1 and the imine of Ix is formed on theax! ax� 1 pathway. The dissociation of this proton-bound dimeris determined by the internal energy distribution of thefragmenting population and the PAs of the imines formed

SCHEME 19.

& PAIZS AND SUHAI

536

(Harrison et al., submitted for publication) and the ax� 1/Ix ionabundance ratio can be approximated by using suitable general-ization of Equation 2. This mechanism is supported by thepresence of weak ion signals corresponding to loss of CO fromthe a2 ions derived from protonated H-Gly–Ala-OH, H-Ala–Gly-OH, and H-Val–Ala-OH. Also, for many of the investigateda2 ions (e.g., a2 of H-Leu–Phe-NH2, H-Phe–Leu-NH2, H-Tyr–Phe-NH2, H-Phe–Tyr-NH2) both the a1 and internal immoniumions are observed with the most abundant being that formed byproton attachment to the monomer of higher PA.

Another route which leads to internal immonium ionsinvolves formation of yz ions which fragment further on thea1� yz pathway as shown in Scheme 21. This mechanism issupported by metastable ion and low-energy collision induceddissociation experiments (Ambihapathy et al., 1997) on variouspeptides including H-Tyr–Gly–Gly-OH, H-Pro–Gly–Gly-OH,H-Phe–Leu-OH, etc. which show energetics characteristic of thea1� yz pathway. It is important to note here that formation of thePhe immonium ion of protonated H-Tyr–Gly–Gly–Phe–Leu-OH can be explained by the [MH]þ! y2! IPhe route (Ambiha-pathy et al., 1997).

A special case occurs if the y1 ion (protonated amino acid)dissociates further to the IN immonium ion by eliminating COand water. This reaction is of importance for tryptic peptidessince interpretation of the corresponding MS/MS spectra is oftenfacilitated by assigning the C-terminus to Lys or Arg using theimmonium ion masses.

F. bx� yz and Diketopiperazine Pathways at Work:Towards Understanding the Proline Effect

Discussing the proline effect postulated for a long time as one ofthe examples of the amino acid selective fragmentation channelswith referring to the non-selective sequence pathways (bx� yz,diketopiperazine, etc.) seems a strange idea at the outset.However, there seems to be a major difference between thechemistries behind the proline and other specific effects (like forexample the aspartic acid and histidine effects). The latter areclearly because of specific chemical activity of the corresponding

side chains, which can be shut down by appropriate chemicalmodifications (e.g., esterification of the Asp side chain). Onthe other hand, it is the personal view of the present authors thatthe proline residues exert their specific activity via affecting theotherwise non-specific fragmentation pathways in such a waythat leads to dominance of a few ions. These preliminaryconsiderations are based on investigations currently under way inour laboratory on a large number of peptides containing proline.We believe that detailed studies on the bx� yz and diketopiper-azine pathways of protonated peptides containing proline willsatisfactorily explain the major characteristics of the prolineeffect.

Many of the protonated proline containing peptides showdistinct fragmentation behavior producing abundant y ions N-terminal to the proline residues. The proline effect (Schwartz &Bursey, 1992) was intestigated by CID studies of pentapeptidescontaining proline at various positions showing that H-Ala–Ala–Pro–Ala–Ala-OH and H-Ala–Ala–Ala–Pro–Ala-OHfragment producing abundant y3 and y2 ions, respectively. Theproline effect was explained by Schwartz & Bursey (1992)considering the high PA of proline.

Vaisar & Urban (1996) have investigated the CID spectra ofanother set of protonated pentapeptides including H-Val–Ala–Pro–Leu–Gly-OH, H-Val–Ala–NMeAla–Leu–Gly-OH, andH-Val–Ala–Pip–Leu–Gly-OH. As expected, protonated H-Val–Ala–Pro–Leu–Gly-OH produces an abundant y3 ionwhereas both protonated H-Val–Ala–NMeAla–Leu–Gly-OHand H-Val–Ala–Pip–Leu–Gly-OH fragment mainly to form b3

ions. The behavior of H-Val–Ala–Pip–Leu–Gly-OH is espe-cially striking since the amino acids Pro and Pip differ only bya –CH2– group. The behavior of H-Val–Ala–NMeAla–Leu–Gly-OH and H-Val–Ala–Pip–Leu–Gly-OH suggests thatmethylation of the amide nitrogen (N-methylation effect)promotes formation of the b ions of the C-terminal neighboramide bond. An explanation of the dramatic difference betweenthe CID spectra of H-Val–Ala–Pro–Leu–Gly-OH and H-Val–Ala–Pip–Leu–Gly-OH cannot be based on PA considerations.To account for this effect, Vaisar & Urban (1996) have proposedthat formation of the b ion on the C-terminal side of Pro is not

SCHEME 20.


537

favored because of the highly strained [3.3.0] bicyclic moietyformed. The corresponding H-Val–Ala–Pip–Leu–Gly-OH b3

ion has a less strained [4.3.0] bicyclic moiety and the N-methylation effect determines the fragmentation. It is worthnoting here that the metastable ion spectrum of protonated H-Gly–Pro–Gly-OH shows a dominant b2 ion (Ambihapathy et al.,1997) which contains the [3.3.0] bicyclic moiety. Since otherlow-energy fragmentation channels leading to y1 ions areavailable for protonated H-Gly–Pro–Gly-OH, statements aboutthe disfavored energetics of the b ion having the [3.3.0] bicyclicmoiety should be considered cautiously. Computational studies(Paizs, unpublished data) on the structure and energetics of

various fragment ions of H-Gly–Pro–Gly-OH led to similarconclusions.

The present authors believe that the proline residue has nospecific effect on the cleavage of the N-terminal amide bond ofprotonated peptides and the cleavage of such amide bonds can bedescribed by the rules of the a1� yz pathway. For example, theresults of metastable ion and low-energy CID studies onprotonated H-Val–Pro-OH (Harrison et al., 2000) are in linewith the basic characteristics of the a1� y1 pathway leading topreference of the y1 ion at low energies (PA of Pro is higher thanthat of the imine derived from Val (Paizs et al., 2004)). Asmentioned above, Pro seems to have a rather specific effect when

SCHEME 21.

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538

located at the C-terminal of tripeptides. For example, protonatedH-Leu–Gly–Pro-OH (Nold et al., 1997) shows a rather specificfragmentation pattern in the sense that the majority of the y1 ionsare formed on the ‘diketopiperazine’ pathway whereas othertripeptides like H-Gly–Gly–Gly-OH, H-Gly–Gly–Leu-OH,etc. produce y1 ions on the b2� y1 pathway.

G. bx� yz, bx!bx�1, and bx! ax Pathways atWork: Fragmentation of Cyclic Peptides

The fragmentation mechanism of cyclic peptides differssignificantly from that of linear peptides. This is because of thefact that there is no free terminal amino group in cyclic peptidesand the peptide ring has to be opened prior to fragmentation.Because of the latter, all sequence ions of protonated cyclicpeptides are originated from secondary cleavages of amidebonds. Protonated cyclic peptides fragment by loss of CO andproducing sequence ions. Sequence ions of cyclic peptides aredenoted by bx

n�m and yzn�m where the superscript defines the

pair of amino acids between which the backbone opening takesplace. In the following, we briefly summarize a fragmentationscheme developed recently (Jegorov et al., 2003) for cyclicpeptides based on the bx� yz, bx! bx� 1, and bx! ax pathways.

Fragmentation pathways of a general cyclic pentapeptideare shown in Schemes 22 and 23. Since cyclic peptides do nothave a free N-terminus, their fragmentation pathways aredominated by the bx� yz channels. The most stable protonationsite (disregarding the basic amino acid side chains) of cyclicpeptides is one of the amide oxygens. Mobilization of the addedproton leads to amide nitrogen protonated species. Ring openingtakes place via oxazolone formation, resulting in a linear peptideion having a free N-terminus and an oxazolone ring at the C-terminus (bx� yz pathway). The linear ion can fragment byloosing CO on the bx! ax pathway, forming a lower b ion(bx! bx� 1 pathway), or by further mobilization of the extraproton to an amide nitrogen and subsequent fragmentation on thebx� yz pathways.

The general pentapeptide shown in Schemes 22 and 23contains a methylated amide nitrogen (aa(3)–aa(4) peptidebond). As noted in the preceding subsection, N-methylationinduces formation of the b ion of the C-terminal neighbor amidebond in linear peptides (Vaisar & Urban, 1998). For the cyclicpentapeptide the corresponding reaction leads to cleavage of theaa(4)–aa(5) amide bond resulting in a linear peptide as shown inScheme 23. The nitrogen of the oxazolone ring of this linearpeptide is methylated, so the ion has a fixed charge. Such ions canfragment further by reactions specific to the oxazolone ring, forexample, by losing CO on the bx! ax pathway and by forming asmaller linear b ion on the bx! bx� 1 pathway (b3

5–4 in thepresent case). It is worth noting here that b3

5–4 has a mobileproton since the fixed charge was eliminated in the last step.

These mechanistic considerations can be easily generaliz-ed to other cyclic peptides. As a case study, we haverecently explained the fragmentation behavior of protonatedRoseotoxin A (cyclo(-Ile-MeVal-MeAla-beta-Ala-2-hydroxy-4-methyl-pentaonyl-3-methyl-Pro-) (Jegorov et al., 2003) based onthese schemes. Some of the mechanistic considerations wereprobed by modeling the fragmentation pathways of protonatedRoseotoxin A and by MS/MS experiments.

III. CHARGE-REMOTE PEPTIDEFRAGMENTATION PATHWAYS

A. The ‘Aspartic Acid’ Effect

Many of the protonated peptides containing aspartic or glutamicacid residues show distinct fragmentation behavior producingabundant b ions C-terminal to these residues. Yu et al. (1993)have investigated the origin of facile cleavages at Asp–Pro andAsp-Xxx peptide bonds by MALDI-TOF MS (Xxx denotes otheramino acids). They have found that the Asp-Pro peptide bond ismore labile than the other peptide bonds regardless of the size ofthe peptide investigated. The key role of the aspartic acid sidechain in the lability of the Asp–Pro peptide bond has beendemonstrated by esterification of the COOH group of the Aspside chain which shuts down dominant formation of the b ion C-terminal to Asp. Bakhtiar et al. (1994) have proved that theactivity of the aspartic acid residue depends on the charge state ofthe ion under investigation. It was found that cleavage of theAsp(75)–Met(76) peptide bond in the a-chain of humanapohemoglobin is observed primarily in the 11þ and 12þionization states of the protein. Qin & Chait (1995) have alsoobserved preferential cleavage of the peptide bonds adjacent toAsp and Glu residues using a MALDI ion trap mass spectrometer.Price et al. (1996) have investigated the 11þ ion of ubiquitinusing BIRD. These authors have found that ubiquitin11þ

fragments nearly exclusively to produce the b527þ/y24

4þ com-plementary ion pair. This specific fragmentation has beenattributed to the Asp(52) residue of ubiquitin.

Selective cleavages at acidic residues including Asp, Glu,and Cys* have been studied by the Wysocki and Gaskell groupson a large number of different peptides (Tsaprailis et al., 1999)under different experimental conditions. The main conclusionsof this work are that nonselective cleavages along the peptidebackbone occur when the number of ionizing protons exceeds thenumber of arginine residues (active bx� yz pathways), whereascleavages adjacent to the acidic residues predominate when thenumber of ionizing protons equals the number of arginineresidues (active selective pathways). The latter observation isexplained by the effective sequestration of the added proton(s) bythe side chain of the arginine residue(s) that makes the otherwiseinactive (energetically disfavored if a mobile proton is present)charge-remote dissociation channels competitive. The charge-remote character of the aspartic acid effect has been proved byCID studies on peptides containing a fixed charge and Aspresidues (Gu et al., 2000) which behave similarly to peptideswhere the number of added protons and the number of arginineresidues are equal.

While our knowledge on the aspartic acid effect was nodoubt enhanced in the last few years, fine energetic and kineticdetails of the underlying processes are still not known. It is nowclear that the COOH group of the Asp side chain takes part in theenhanced amide bond cleavage but the mechanism is stilluncertain. Also, there are two main possibilities for the structureof the ions showing pronounced aspartic acid effect. Because theside chain of Arg contains a functionality of high PA, it isassumed that these groups are nearly always protonated under themost common experimental conditions. Therefore, the mostimportant structure-determining factor for these species is


539

‘‘internal solvation’’ or ‘‘CS’’ of the protonated Arg side chain(s)by the various electron rich groups in the rest of the molecule.Carboxylic groups in side chains of acidic residues, the terminalamino group, and the amide oxygens function as electron-donors;their role is simply solvation of the protonated basic moiety of theion by H-bonding. If the acid–base interactions are strongenough, formation of a SB can take place (Yu et al., 1993; Priceet al., 1996; Tsaprailis et al., 1999) with simultaneous transfer ofthe acidic proton of the acidic side chain to other functionalgroups of the molecule. The nature of these interactions depends

on the amino acids involved, size of the peptide, internal energyof the ions formed, etc. The mechanisms proposed to account forthe aspartic acid effect involve both CS and SB structures atvarious phases of the fragmentation.

The mechanism proposed by Yu et al. (1993) to account forthe aspartic acid effect is shown in Scheme 24a (Path A, compiledfor the case of a general peptide containing both Asp and Arg). Itis assumed that the ‘‘ionizing’’ proton added to the system issequestered by the arginine (basic) side chain. In the first step, theacidic proton of COOH transfers to the nitrogen of the amide

SCHEME 22.

& PAIZS AND SUHAI

540

bond adjacent to the aspartic acid (Asp) residue forming theintermediate which contains a SB. Because of the excess energydeposited on the ion during its formation and excitation, theAsp-Xxx bond cleaves forming a cyclic anhydride. Price et al.(1996) have proposed a slightly different mechanism (Path B,Scheme 24b). The first step of this mechanism is again salt bridgeformation between the carboxylate side chain of aspartic acid andthe nitrogen of the adjacent amide bond (i.e., a proton transferfrom the aspartic acid side chain to the neighbor amide nitrogen).The only difference between the first steps of Paths A and B is thatPrice et al. (1996) assume a protonated arginine side chain nearbystabilizes the SB making it a long-lived intermediate whereas Yu

et al. (1993) contrary to the fact that their peptides also containprotonated Arg side chains, do not consider such an effect. In thesecond step of Path B direct bond cleavage of the amide bondadjacent to the aspartic acid residue occurs. BIRD experiments(Price et al., 1996) indicate that the rate limiting step of a possiblecomplex reaction scheme has a very high frequency factor. Thisfurther suggests an entropically highly favored mechanism suchas a direct bond cleavage of the amide bond adjacent to theaspartic acid residue. Also, Path B assumes that a relatively longliving intermediate—the SB formed by the side chain of Asp(52)and the protonated nitrogen of the adjacent amide bond—isformed in a faster reaction than the dissociation of the SB species.

SCHEME 23.


541

SCHEME 24.

& PAIZS AND SUHAI

542

SCHEME 24. (Continued )


543

A weakness of Path B is that direct bond cleavage of thenitrogen protonated amide bond leaves behind an extremelyreactive acylium ion. It is clearly not likely that such a reactivemoiety would survive in a large protein. Computational studiesand MS/MS experiments on protonated H-Arg–Asp-NH2 (Paizset al., 2002) indicate that another pathway (Path C, Scheme 24c)operates, where instead of the direct bond cleavage, the Asp-Xxxpeptide bond is cleaved on the corresponding bx� yz pathway,that is by nucleophilic attack of the N-terminal neighbor amide

oxygen on the carbon center of the protonated amide bond. Thisreaction leads to a b ion with classical oxazolone structure.

A common weakness of mechanisms proposed to accountfor the aspartic acid effect based on SB species is that selectivityof the chemistry is hardly explained. This is because of the factthat if a SB is formed, the by-product mobile proton (COOHproton transferred to backbone) could very probably ‘‘visit’’various backbone protonation sites inducing random fragmenta-tion at the backbone amide bonds. A mechanism that involves

SCHEME 25.

& PAIZS AND SUHAI

544

only charge solvated species has been recently proposed(Tsaprailis et al., 1999; Paizs et al., 2002). The low-energy CSspecies contain many very flexible torsion angles. For example,the side chain of aspartic acid can rotate rather freely wheneverthe carboxylic group is not involved in solvation of the protonatedguanidine moiety. During its rotation, the hydroxyl group can getclose to the carbon center of theC-terminal neighbor amide bond.Thus, simultaneous ring formation and transfer of the acidicproton to the amide nitrogen can occur via a four-center-one-stepmechanism. Such a reaction (Path D, Scheme 24d) results in theformation of a cyclic anhydride and cleavage of the attackedamide bond. The attack of the hydroxyl oxygen of thecarboxylate group on the amide carbon is entropically favoredsince the reaction results in a five-membered species.

Contrary to recent developments, energetic, kinetic, andentropy factors determining the activity of the aspartic acidchannels are still not yet fully known. The mechanisms describedabove (Path A–D) must be further probed on peptides containingAsp, varying the length, and the primary sequence of the peptide.The time-scale of the experiments seems to be also a criticalfactor; differences between the activities of Asp and Glu dependheavily on instrumental setup.

B. Loss of CH3SOH From the Side Chain of OxidizedMethionine Residues

Methionine oxidation is one of the most frequently occurringmodification to peptides, which is mostly caused by gel electro-phoresis sample preparation. The identification of peptidescontaining oxidized methionine is facilitated by the character-istic loss of methane sulfenic acid (CH3SOH, 64 Da). Loss ofCH3SOH occurs most dominantly in singly protonated trypticpeptides indicating that the corresponding elimination reaction isa charge-remote process. The mechanism of the elimination ofCH3SOH from oxidized methionine has been investigated byO’Hair & Reid (1999) utilizing MS/MS/MS and deuteriumlabeling experiments. Charge directed elimination of CH3SOH(Scheme 25, route 1) is initiated by mobilization of the extraproton to form CH3-SOHþ- which is then eliminated by nuc-leophilic attack of either the Xxx-Met or the Met-Xxx amideoxygen, leading to formation of a six- or five-membered ring,respectively. Alternatively, a charge remote mechanism involveselimination of CH3SOH via 1,2-cis-elimination from the neutralside chain (Scheme 25, route 2). The labeling and MS/MS/MSdata suggest that the charge remote mechanism is highly com-petitive with the charge directed PFPs explaining the dominantloss of CH3SOH from oxidized methionine containing singlycharged trytic peptides.

IV. CONCLUDING REMARKS

This review attempted to summarize the dissociation chemistryof protonated peptides paying special attention to classificationand characterization of fragmentation pathways leading tostructurally valuable sequence non-sequence ions. It has beenshown that the ‘mobile proton’ model of peptide fragmentationcan be used to understand the MS/MS spectra of protonatedpeptides only in a qualitative way rationalizing differences

observed for the low-energy fragmentation of peptide ionshaving and lacking a mobile proton. To meet all the demands setforth by the exploding field of proteomics, deeper understandingof the dissociation chemistry of protonated peptides is needed. Tothis end the PIC model that is based on interplay of the majorfragmentation pathways of protonated peptides is proposed.These fragmentation pathways can be fully characterized toinclude all the pre-dissociation, dissociation, and post-dissocia-tion events involved leading to semi-quantative understandingthe MS/MS spectra of protonated peptides. Experimentaland computational data on the fragmentation of protonatedpeptides are reevaluated in the light of the PIC model and themajor PFPs.

While some of the PFPs are characterized in quite anadvanced manner there is still a lot to study in the chemistry ofpeptide fragmentation. To develop robust IIRs of the MS/MSspectra of protonated peptides, further investigations are needed.These studies will involve both the ‘bottom up’ chemical and ‘topdown’ statistical approaches. The present authors’ opinion is thatwhereas the ‘bottom up’ approach will provide the necessaryframework (data model) via the PIC model of peptide frag-mentation, statistical approaches will be applied to derive theprimary structure related quantitative parameters to be used in theIIRs.

ACKNOWLEDGMENTS

BP thanks Prof. Alex G. Harrison, Prof. Chrys Wesdemiotis,Dr. Karoly Vekey, Dr. Arpad Somogyi, Dr. Martina Schnolzer,Dr. George Lendvay, and Dr. Istvan Csonka for many usefuldiscussions on the chemistry of protonated peptides.

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Bela Paizs received his M.S. and Ph.D. degrees in Chemistry from the Eotvos University

(Budapest) in 1992 and 1998. Since 1997 he is a postdoctoral fellow at the German Cancer

Research Center in Heidelberg, Germany. His research is mainly focused on protein mass

spectrometry, bioinformatics, theoretial chemistry, kinetics of chemical reactions, and

applied numerical analysis.

Sandor Suhai is head of the Department of Molecular Biophysics at the Deutsches

Krebsforschungszentrum in Heidelberg. He studied physics, mathematics, and chemistry in

Budapest, Gottingen, and Erlangen, respectively, and holds a Ph.D. in theoretical physics

from the Roland Eotvos University, Budapest, and in theoretical chemistry from the

Friedrich-Alexander University, Erlangen, where he received his habilitation in theoretical

chemistry in 1984. He was habilitated in molecular bioinformatics at the Ruprecht-Karls

University, Heidelberg, in 1986 where he became a professor in 1998. His research interests

have concentrated on computer-aided modeling of biomolecular phenomena, including

molecular mechanical and dynamical simulations of intra- and intermolecular interactions

in DNA and proteins, the mathematical and information-theoretical analysis of their

sequences, and the development of new quantum-theoretical methods to treat various

physical and chemical properties of biopolymers. In addition, he has devoted considerable

attention to the bioinformatics aspects of genome research and is involved in projects aimed

at functional analysis of the human genome.

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Fragmentation in MSMS