FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
Béla Paizs1,2* and Sándor Suhai1
1
Department of Molecular Biophysics, German Cancer Research Center,
Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany
2
Protein 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 reviewed
in the present paper paying special attention to classification of
the known fragmentation channels into a simple hierarchy
defined according to the chemistry involved. It is shown that the
‘mobile proton’ model of peptide fragmentation can be used to
understand the MS/MS spectra of protonated peptides only in a
qualitative manner rationalizing differences observed for lowenergy collision induced dissociation of peptide ions having or
lacking a mobile proton. To overcome this limitation, a deeper
understanding of the dissociation chemistry of protonated
peptides is needed. To this end use of the ‘pathways in
competition’ (PIC) model that involves a detailed energetic
and kinetic characterization of the major peptide fragmentation
pathways (PFPs) is proposed. The known PFPs are described in
detail including all the pre-dissociation, dissociation, and postdissociation events. It is our hope that studies to further extend
PIC will lead to semi-quantative understanding of the MS/MS
spectra of protonated peptides which could be used to develop
refined bioinformatics algorithms for MS/MS based proteomics.
Experimental and computational data on the fragmentation of
protonated peptides are reevaluated from the point of view of the
PIC model considering the mechanism, energetics, and kinetics
of the major PFPs. Evidence proving semi-quantitative predictability 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,
2005
Keywords: protonated peptides; peptide fragmentation; reaction mechanism; mass spectrometry
I. INTRODUCTION
A. Protein Sequencing by Mass Spectrometry (MS)
MS has become an important tool for determining the amino acid
sequence of peptides and proteins. The MS technique involves
creation and detection of charged peptide and protein ions in the
gas phase. Soft ionization techniques like electrospray ionization
(ESI, Fenn et al., 1989) and matrix-assisted laser desorption/
ionization (MALDI, Karas & Hillenkamp, 1988) are used to
produce intact peptide and protein ions in the gas phase (mostly in
————
*Correspondence to: Béla Paizs, Protein Analysis Facility, Deutsches
Krebsforschungszentrum, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. E-mail: B.Paizs@DKFZ.de
Mass Spectrometry Reviews, 2005, 24, 508– 548
# 2004 by Wiley Periodicals, Inc.
positive ion mode, e.g., via protonation) without fragmentation.
The mass to charge ratio (m/z) of these ions can be rapidly and
accurately measured allowing such applications like fast evaluation of the correctness of the sequence of peptides and proteins,
checking the presence of post-translational modifications, and
application of bioinformatics-assisted peptide-mass fingerprinting (PMF, Henzel et al., 1993; James et al., 1993; Mann, Hojrup,
& Roepstorff, 1993; Pappin, Hojrup, & Bleasby, 1993; Yates
et al., 1993) methods. Analysis by using PMF is based on
digestion of the protein with a site-specific enzyme (mostly
trypsin) and comparison of the measured peptide molecular
weights (peptide mass fingerprint) to those predicted in silico for
the sequences in protein and/or translated nucleic acid databases.
Provided that a sufficient number of peptide ions are observed in
the MS experiment and the protein is not heavily modified, a
match can be generally found.
The power of this bioinformatics based strategy can be
dramatically increased by employing methods of tandem mass
spectroscopy (McLafferty, 1983). In the tandem MS (MS/MS)
and MSn experiments, the first mass analyzer is used to
selectively pass an ion into another reaction region where
excitation and dissociation take place. The second mass analyzer
is used to record the m/z values of the dissociation products. (MS/
MS experiments in the quadrupole ion trap or Fourier transform
ion cyclotron instruments are tandem in time and use the same
volume for the above processes.) Excitation of the precursor ion
is most commonly achieved by energetic collisions with a nonreactive gas, such as argon or helium, and is referred to as
collision-induced (activated) dissociation (CID or CAD).
The observed fragmentation pattern depends on various
parameters including the amino acid composition and size of the
peptide, excitation method, time scale of the instrument, the
charge state of the ion, etc. Peptide precursor ions dissociated
under the most usual low-energy collision conditions fragment
along the backbone at the amide bonds (Hunt et al., 1986;
Biemann, 1988; Papayannopoulos, 1995) forming structurally
informative sequence ions and less useful non-sequence ions by
losing small neutrals like water, ammonia, etc. The sequence ions
involve 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 of
protonated peptides can be used to identify proteins in several
ways. Amino acid residues can be determined from the mass
difference of successive fragment ions of the same type (e.g., bn
and bn 1). In this way, one can apply tandem MS for even de novo
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
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SCHEME 1.
peptide sequencing (Dancik et al., 1999; Taylor & Johnson, 2001)
provided the corresponding ion series are present in the MS/MS
spectrum. Some algorithms make use of MS/MS data to generate
‘peptide-sequence tags’ (Mann & Wilm, 1994) that consist of
information on a short stretch of sequence. Another approach is
based on in silico generation of MS/MS fragmentation patterns
(Eng, McCormack, & Yates, 1994; Clauser, Baker, & Burlingame, 1999; Perkins et al., 1999) for peptides derived from
entries of protein and translated nucleic acid databases and
comparison of the predicted spectra to those determined
experimentally.
It is evident from this short introduction that the level of our
understanding of the main fragmentation processes of protonated
peptides is critical not only from the academic point of view but
also for practical reasons. For example, deriving ‘peptidesequence tags’ from the MS/MS spectra requires some knowledge of the major fragmentation pathways and rules. Since
protonated peptides dissociate on a large number of different
fragmentation pathways, generation of in silico MS/MS spectra
considering both the ion m/z and fragment ion abundance
dimensions for general peptide entries of databases is an extremely difficult task. Also, some peptides show selective and/or
enhanced fragmentation (Wysocki et al., 2000) at some of the
amino acid residues producing MS/MS spectra poor in valuable
sequence ions. In the light of these facts, it is not surprising that
existing sequencing programs use only the information inherent
in the m/z values of the most important sequence ions and discard
any ion intensity-related data. Protein identification, using
tandem MS could be no doubt further developed if the major
rules deriving the fragmentation of protonated peptides were
known to such an extent that would permit predictions of some of
the fragment ion intensity relationship (IIRs) of the MS/MS
spectra of protonated peptides.
There are two major ways to determine IIRs for the MS/MS
spectra of protonated peptides. The first, a ‘top down’ strategy
pioneered by Wysocki, Yates, and Simpson (Huang et al., 2002;
Kapp et al., 2003; Tabb et al., 2003) is a statistical approach based
on systematic assessment of large databases containing MS/MS
spectra of protonated peptides to derive fragmentation rules. The
second, a ‘bottom up’ chemical approach involves systematic
investigation of the major fragmentation pathways of protonated
peptides to increase our knowledge on the dissociation chemistry
involved. The personal view of the present authors is that new
highly efficient peptide sequencing algorithms utilizing MS/MS
IIRs for protonated peptides will be based on results delivered by
intensive interplay of the ‘top down’ statistical and the ‘bottom
up’ chemical approaches.
The present paper reviews the dissociation chemistry of
protonated peptides by paying special attention to recent
developments that could be used to explain and in some extent
to predict MS/MS IIRs.
B. Mobile Proton Model
The most comprehensive model currently available to describe
how protonated peptides dissociate upon excitation is termed the
‘mobile proton’ model. This has emerged as a result of a large
number of studies performed by Wysocki (Jones et al., 1994;
Dongré 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 collision
conditions 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, peptides
can be protonated at various protonation sites (terminal amino
group, amide oxygens and nitrogens, side chain groups) leading
to various isomers. There are two major classes of peptide ions,
which differ in the energetics of the isomers produced by
protonation. In the first class, one or more of the protonation sites
is energetically and/or kinetically more favored than the others
leading to sequestration of the added proton(s), for example,
singly protonated tryptic peptides containing arginine at the Cterminus. Characteristic of this peptide ion class is the large
energy that is needed to mobilize the extra proton(s) to
energetically less favored protonation sites. For the second
group, many of the protonation sites are accessible in a narrow
energy range. This class is best represented by the practically
important doubly charged tryptic peptides.
As the internal energy of the ions increases upon excitation,
energetically less favored protonation sites like those of the
reactive intermediates leading to backbone dissociation can
become more populated or in the case of hard proton se509
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PAIZS AND SUHAI
questering, charge-remote fragmentation (e.g., ‘aspartic acid’
effect, see later) can occur. Considering the reactive intermediates of the backbone fragmentation, it is known from the results
of molecular orbital calculations (McCormack et al., 1993;
Somogyi, Wysocki, & Mayer, 1994) that protonation on the
amide nitrogen leads to considerable weakening of the amide
bond, whereas protonation on the amide oxygen makes the amide
bonds even stronger than those in the neutral species. On the other
hand, protonation on the amide nitrogen is not thermodynamically favored compared to protonation on amide oxygens, or on
the N-terminal amino group or basic amino acid (AA) side chains
such as those of arginine and lysine. In short, from the point of
view of decomposition, protonation on the amide nitrogen is
favorable, whereas from the thermodynamic point of view this
site is not the most favored one. The ‘mobile proton model’
introduced by Wysocki (Dongré et al., 1996) and co-workers
resolves this discrepancy by stating that upon excitation the
proton(s) added to a peptide will migrate to various protonation
sites prior to fragmentation provided they are not sequestered by
a basic amino acid side chain. Essentially the same concept has
been developed in Gaskell’s group (Burlet, Yang, & Kaskell,
1992) by introducing the ‘heterogeneous population model’ that
assumes the existence of different protonated forms for easily
fragmenting species and the dominance of a single structure for
those peptide ions in which the added proton is sequestered.
The ‘mobile proton’ model has been verified by using
deuterium labeling techniques (Tsang & Harrison, 1976;
Mueller, Eckersley, & Richter, 1988; Johnson, Krylov, & Walsh,
1995; Harrison & Yalcin, 1997) which indicate strong H/D
mixing prior to collisionally activated dissociation of [M þ D]þ
ions of a number of amino acids and small peptides. In a very
early study, Tsang & Harrison (1976) have shown that facile H/D
mixing occurs in the D2 and CD4 chemical ionization of amino
acids. This work was later extended (Harrison & Yalcin, 1997) to
small peptides lacking arginine, the [M þ D]þ ions of which
show high proton mobility between the terminal amino group and
amide Ns and Os. Mueller, Eckersley, & Richter (1988) have
investigated the fragmentation of the [M þ H]þand [M þ D]þ
ions of H-Phe–Phe–Phe-OH and found that practically complete
exchange of the added deuteron with labile hydrogens occur upon
excitation. Johnson, Krylov, & Walsh (1995) have studied the
fragmentation reactions of singly deuterated peptides and
have found that the deuteron is redistributed amongst the
exchangeable sites upon excitation to induce fragmentation of
the parent ion.
Wysocki and co-workers have demonstrated (Jones et al.,
1994; Dongré et al., 1996; Wysocki et al., 2000) that the relative
positions of fragmentation efficiency curves obtained by ESI in
combination with surface induced dissociation (ESI/SID) depend
on the amino acid composition (absence or presence and type of a
basic residue) and on the sequence and the size of the peptide
investigated. These studies have benefited from the relatively
narrow internal energy distribution of SID-generated ions and the
fact that the average energy of the ion population can be easily
changed. Investigation of a large number of peptides with
systematically changed amino acid composition showed that
backbone dissociation of protonated peptides under low-energy
conditions is a charge-directed process. Also, the energy required
for proton ‘mobilization’ from a basic side chain or the amino510
terminus depends on the amino acid composition, with dissociation energy requirements greatest for arginine-containing peptides and decreasing in the order of Arg-containing > Lyscontaining > non-basic peptides, mimicking the order of
decreasing gas-phase basicity. In selected cases, more energy
might be required to mobilize the sequestered proton than is
required to initiate charge-remote fragmentation pathways
(aspartic acid effect, see later). Mechanistic considerations
involved in the ‘mobile proton model’ have been refined by the
Wysocki and Gaskell groups (Tsaprailis et al., 1999) to such an
extent that allows qualitative understanding of the fragmentation
behavior of peptides containing a few arginines and/or acidic
residues and/or protons. Also, the ‘mobile proton’ model was
successfully applied to explain the charge state dependent
fragmentation of gaseous protein ions (Engel et al., 2002). In
those cases, when the number of ionizing protons is larger than
the number of arginines, non-selective fragmentation is observed. If the number of arginines is larger or equal to the number of
ionizing protons, selective fragmentation via the aspartic acid
effect is expected.
The ‘mobile proton’ model has also been validated by using
theoretical tools (Csonka et al., 2000, 2001; Paizs et al., 2001).
Using quantum chemical techniques, the structure and energetics
of various protonated forms of model peptides were determined.
These species are connected by transitions like internal rotations,
proton transfer reactions, etc. After determining the structures
and energetics of the corresponding transition structures, one can
apply quantum chemical data (like relative energies and
vibrational frequencies) in the RRKM formalism to approximate
the unimolecular reaction rates and the time scale of the
underlying processes. By collecting all these data, it is possible
to construct ‘proton traffic maps’ (Csonka et al., 2000) that
contain the energy required to mobilize the added proton between
various protonation sites including the terminal amino group,
amide Ns and Os, and amino acid side chains. These theoretical
investigations unequivocally proved that the energy requirements for mobilizing the added proton increase in the N-formylglycineamide (no terminal amino group, transitions only between amide oxygens and nitrogens, Csonka et al., 2000), HGly–Gly-OH (Paizs et al., 2001), and H-Lys–Gly-OH (Csonka
et al., 2001) series in agreement with the ‘mobile proton’ model.
For the investigated systems, it was shown that the added proton
can sample all protonation sites prior to fragmentation at internal
energies well below the threshold energy of the most favored
fragmentation 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/MS
spectra of protonated peptides depends on two major factors
which include the probability for the cleavage of the corresponding amide bond and mechanistic aspects that decide which fragment will keep the added proton(s) during the spatial separation
of the products. The dissociation probability depends on the
energetic and kinetic accessibility of the reactive configurations
and on the actual rate constants of the bond cleavages themselves.
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
In most of the cases, the fate of the separating fragments is
decided by the thermodynamics involved, that is, the fragment
with larger proton affinity (PA) will usually keep the added proton
responsible for the charge-directed dissociation. In other words,
formation of fragment ions of protonated peptides involves predissociation (proton transfer reactions, transitions between
isomers and tautomers, cis–trans isomerization of amide bonds,
etc.), dissociation, and post-dissociation events. One can fully
understand the MS/MS spectra of protonated peptides only if the
major mechanistic, energetic, and kinetic aspects of the predissociation, dissociation, and post-dissociation processes are
known.
In the following, we briefly evaluate the ‘mobile proton
model’ of peptide fragmentation considering the very general
points described in the previous paragraph. Following the
introduction of ‘soft’ ionization techniques like ESI and MALDI
(Karas & Hillenkamp, 1988; Fenn et al., 1989) and the general
availability of triple quadrupole and ion trap instruments, the late
1980s and early 1990s saw an unambiguous shift in MS/MS
peptide analysis from the high to the low fragmentation-energy
regime. The high-energy MS/MS spectra of protonated peptides
contain ions formed on both backbone and charge-remote
fragmentation pathways (Papayannopoulos, 1995). The latter
reactions do not correlate with the position of the ionizing proton
on the peptide chain. The majority of the high-energy
fragmentation pathways are initiated by reactions involving
direct bond cleavages. Such reactions are known to be facile only
if the ion population is significantly excited. Changing the
excitation conditions to favor the low fragmentation-energy
regime made the high-energy charge-remote dissociation
channels inaccessible since the majority of the ions excited
under low-energy collision conditions do not have enough energy
to fragment in reactions involving direct bond cleavages at
observable rates. However, protonated peptides have proved to
dissociate in the low fragmentation-energy regime efficiently.
The major success of the ‘mobile proton model’ was to provide a
solid background to qualitatively understand the underlying
chemistry (i) by proving direct involvement of the ionizing
proton in the majority of the low-energy fragmentation processes
(charge-directed pathways), (ii) by showing the lability of
nitrogen protonated amide bonds, and (iii) by explaining how
selective charge-remote processes (e.g., aspartic acid effect, see
below) can become competitive if the mobility of the added
proton is dramatically decreased by sequestration of strongly
basic amino acid side chains.
Contrary to the many successful attempts used to qualitatively explain the MS/MS spectra of various protonated peptides,
the ‘mobile proton’ model considers only pre-dissociation
processes of peptide fragmentation discussing mainly accessibility or inactivity of proton-transfer pathways which lead to
reactive intermediates of sequence ion fragmentation channels.
For example, making a distinction between active and inactive
proton transfer pathways is enough to explain why doubly
charged tryptic peptides containing Arg at the C-terminus
fragment more easily than the corresponding singly charged
species. However, the ‘mobile proton’ model is not able to answer
such simple questions, as to why the low-energy CID spectra of
protonated Leu-enkephaline is dominated by loss of the Cterminal Leu residue or why many of the fragment ions expected
&
to be present in the MS/MS spectra of doubly charged tryptic
peptides do not appear at all.
As mentioned above clear understanding of the fragmentation pathways and the key factors determining the MS/MS ion
abundances of protonated peptides is needed to improve the
existing bioinformatics based protein identification tools. Over
the past years, there has been considerable activity to provide
more detailed information on the ion structures and fragmentation pathways of protonated peptides. However, both the ‘bottom
up’ chemical and ‘top down’ statistical approaches are still in
their infancy. The first statistical evaluations of the MS/MS data
of a large number of protonated peptides are just appearing
(Huang et al., 2002; Kapp et al., 2003; Tabb et al., 2003) and there
is still a lot to understand in the dissociation chemistry of
peptides. There is also no doubt that the ‘bottom up’ chemical
strategy has to go far beyond the ‘mobile proton’ model which
describes only pre-dissociation events of peptide fragmentation.
A new—more powerful—model is needed which could be used
to predict at least some of the IIRs of the MS/MS spectra of
protonated peptides. To do so, the new model has to involve at
least a semi-quantitative description of all pre-dissociation,
dissociation, and post-dissociation processes of the most
important fragmentation pathways.
In the following, we outline the major characteristics of the
pathways in competition (PIC) model of peptide fragmentation
by analyzing the dissociation chemistry involved in the different
fragmentation pathways. This model is based on classification of
the PFPs into a simple hierarchy; mechanistic, energetic, and
kinetic description of the individual pathways; and a kinetic
approach used to describe competition of the various pathways. It
is to be noted here that the PIC model summarizes the results of a
large number of studies performed in the Boyd, Bursey, Harrison,
Gaskell, Glish, O’Hair, Paizs, Vaisar, Wesdemiotis, Wysocki, and
co-workers. We do believe that in the near future PIC will provide
a flexible, concise, and powerful framework that will be used in
the ‘top down’ statistical approach as a data model to determine
MS/MS IIRs for improved protein identification tools.
1. Pathways in Competition Model
of Peptide Fragmentation
Dissociation of protonated peptides can be described as a
competition between charge-remote and charge-directed PFPs in
a complicated reaction pattern where fragment ions are formed
with substantially different probabilities. PFPs can be classified
according to a hierarchy shown in Scheme 2. Protonated peptides
in the low fragmentation-energy regime dissociate mainly on
charge-directed pathways. The only low-energy charge-remote
PFPs correspond to the selective cleavage observed for some of
the Asp containing peptides (aspartic acid effect, for details see
below) and side chain reactions of oxidized methionine. The
charge-directed PFPs can be further classified as sequence or
non-sequence dissociation channels. The former produce ions
containing information on the primary structure of peptides
whereas the latter correspond to losses of small neutrals like
water, ammonia, etc. The structurally most valuable b and y
fragment ions of protonated peptides are primarily formed on
sequence PFPs by cleavage of the amide bond. The PFPs leading
511
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PAIZS AND SUHAI
SCHEME 2.
to b and y ions involve migration of the added proton from the
energetically most preferred protonation site to amide nitrogens.
Protonation of the amide nitrogen has two profound effects: (1) it
weakens the amide bond; (2) the carbon atom of the protonated
amide group becomes a likely target of a nucleophilic attack of
nearby electron-rich groups.
Amide nitrogen protonated species can dissociate by direct
bond cleavage, for example, for the N-terminal amide bond of
underivatized protonated peptides on the a1 yx pathway. However, the low fragmentation-energy regime disfavors dissociation
via direct bond cleavage because these reactions are facile only if
large energy is deposited into the ions. Therefore, the majority of
the amide bonds are cleaved in more complex rearrangementtype reactions involving nucleophilic attack of various functionalities on the carbon center of the protonated amide bon. These
functionalities involve either (i) the oxygen of the N-terminal
neighbor amide bond (bx yz pathway) or (ii) the nitrogen of the
N-terminal amino group (aziridinone and diketopiperazine
pathways) or (iii) side chain nucleophiles. The b and y ions
formed on the primary sequence PFPs can fragment further to
form 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 factors
determining the MS/MS spectra of protonated peptides.
For all the low-energy PFPs described above, the fate of the
separating fragments is decided by the thermodynamics involved,
that is, the fragment with larger PA will usually keep the added
proton. The only exceptions are those cases where the fragmentation
introduces chemical changes, which lead to a fixed charge (e.g., see
discussion below on dissociation of peptides containing Nmethylated amide bonds). It is worth noting here, that empirical
formulas have already been derived for some of the PFPs (bx yz
and a1 yx) which allow prediction of some MS/MS IIRs
considering the thermodynamics of the separation of the fragments.
512
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 the
corresponding dissociation channels. However, a more complete
understanding of the MS/MS spectra of protonated peptides
requires the introduction and integration of kinetic models of the
underlying complex reaction pattern.
In the following, experimental and theoretical tools used to
characterize PFPs are briefly discussed. In the main body of the
paper, the chemistries of the various PFPs classified in the
hierarchy of Scheme 2 are critically reviewed.
D. Experimental Tools for Investigating Peptide
Fragmentation Pathways
The experimental tools that can be applied to probe the mechanistic, energetic, and kinetic details of PFPs involve wellestablished techniques of MS like MSn experiments, comparison
of the MS spectra of fragment ions with those of well-defined
reference compounds synthesized independently, exploring the
neutrals 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 not
describe these techniques here; the reader is referred to the
discussion of the various PFPs for possible applications.
A few techniques can provide especially useful information
on 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 ion
abundances and the PA of the constituting amino acids in series of
protonated di- and tri-peptides. For some of the peptides series
investigated, a linear free energy relationship was found
indicating a direct dependence between thermochemistry and
the kinetically determined logarithms of relative ion abundances.
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
Energy-resolved CID and SID studies can be used to probe
energetic details of the PFPs. Klassen & Kebarle (1997) applied
energy-resolved CID-MS to determine the dissociation threshold
energies of fragment ions of various protonated peptides. The
energetics and the dynamics of the primary fragmentation
pathways of small peptides were determined from the RRKM
modeling of the collision energy-resolved fragmentation efficiency curves (Laskin, Denisov, & Futrell, 2000). Hanley and coworkers (Lim et al., 1999) developed a method for determination
of dissociation energies using SID techniques.
Blackbody infrared radiative dissociation (BIRD) developed by Price, Schnier, & Williams (1996) and Dunbar &
McMahon (1998) can also be used to obtain activation energies
and dynamics of dissociation processes of medium-sized and
large biomolecule ions. With BIRD, trapped ions are activated by
interaction with the blackbody radiation field inside the vacuum
chamber of a Fourier-transform mass spectrometer at very low
pressures. From the temperature dependence of the unimolecular
rate constants, Arrhenius activation parameters can be obtained
(Schnier et al., 1997).
E. Theoretical Tools for Investigating Peptide
Fragmentation Pathways
Studying the energetics and kinetics of PFPs by means of
theoretical methods requires special modeling strategies and
involves scanning of the potential energy surface (PES) of
protonated peptides, determining transition structures belonging
to pre-dissociation and dissociation events and the determination
of thermochemical quantities (proton affinities) of final products.
Scanning the PES of protonated peptides is important to
probe the energetics of the different protonations sites, charge
solvated and salt-bridge (SB) forms, etc. (Csonka et al., 2000;
Paizs et al., 2002). A generally used computational strategy to
obtain low-energy structures of peptides is based on molecular
dynamics simulations using protein force fields. Direct application of the available modeling strategies for the case of
protonated peptides is not straightforward since some of the
atom types (protonated amide nitrogen and oxygen) are missing
from the protein force fields used nowadays. This shortcoming
can be circumvented by running the dynamics calculations on
neutral species and protonating a collection of neutral structures
in the second step. The disadvantage of this method is that a large
number of such protonated species have to be further refined as
neutral and protonated species can differ significantly. A better
approach is to derive the missing parameters and run the
dynamics calculations using the extended protein force fields.
Our laboratory is currently involved in a project aimed to define
parameters for protonated amide nitrogen and oxygen atoms. Our
experience shows that even the best structures derived from
dynamics runs have to be refined by quantum chemical
calculations which should be performed at various levels starting
with less accurate models and finishing with density functional
theory (DFT) computations on the most interesting species.
Since MS/MS ion abundances are determined by the kinetics
of the PFPs, transition structures belonging to the investigated
fragmentation pathways, proton transfer reactions, intramolecular rearrangements, etc. have to be searched for. It is worth noting
here that one can obtain reasonable results using only moderate
&
basis sets in conjunction with DFT for the chemistry of
protonated peptides. This is mainly because of the facts that the
positive charge of the ion forces the electrons close to the nuclei
and the most important chemical changes are heterolytic bond
cleavages, which can be described at the DFT levels reasonably.
Efficient data handling in the PES scan phase of the modeling and
the relative ‘simplicity’ of the chemistry involved, allow
researchers to perform detailed studies on protonated oligopeptides having up to 6–7 amino acid residues.
Using the results of the quantum chemical calculations, the
rate coefficients for the transitions between the minima on the
PES can be calculated using the RRKM method (Forst, 1973;
Holbrook, Pilling, & Robertson, 1996). Our experience shows
that the RRKM calculations are particularly useful to predict the
time-scale of the various processes occurring for protonated
peptides because the currently used mass spectrometers cover an
extremely wide range (109 –103 sec) of reaction times.
F. Nomenclature
Throughout the present paper, we refer to fragment ions of
protonated peptides using the nomenclature (Scheme 1) developed by Roepstorff & Fohlmann (1984) and modified by
Biemann (1988). The dissociation mechanisms sometimes
depend on the location of the cleaved amide bond along the
peptide backbone. Various amide bonds are denoted by
specifying the amino acid (aa) residues connected by the amide
bond, that is, aa(1)–aa(2), aa(2)–aa(3), . . . , aa(n)–aa(n þ 1) refer
to the N-terminal first, second, . . . ,nth amide bond in a general
aa(1)–aa(2)–aa(3) . . . aa(n) . . . peptide (containing N amino acid
residues), respectively. Amino acid residues are denoted by their
three-letter code. Some of the PFPs are referred as specific or
non-specific in the text. By the former, we note pathways
involving chemistry determined by a particular amino acid side
chain moiety whereas the later is determined purely by the
interaction of backbone atoms.
II. CHARGE-DIRECTED PEPTIDE
FRAGMENTATION PATHWAYS
A. Dissociation of the aa(n)–aa(n+1) (n>1) Amide
Bond of Protonated Peptides
According to the general scheme described in Introduction of the
present article, protonated amide bonds can be cleaved either by
rearrangement-type reactions or by direct bond cleavage. Under
low-energy collision conditions most of the backbone cleavages of
protonated peptides occur in reactions which belong to the former
class (the only known exception is the a1 yx pathway discussed
below). This is mainly because the excited ions do not have enough
energy to fragment on direct bond cleavage pathways, which are
facile, only if significant energy is deposited into the ions. It is,
therefore, not surprising that the majority of the sequence ions of
protonated peptides are formed on unspecific fragmentation
pathways where either the N-terminal neighbor amide oxygen
(bx yz pathway, Paizs & Suhai, 2002a) or the nitrogen of the
N-terminal amino group (diketopiperazine pathway, Cordero,
513
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PAIZS AND SUHAI
Houser, & Wesdemiotis, 1993) attacks the carbon center of the
protonated amide bond to induce dissociation.
1. bx yz Peptide Fragmentation Pathways
The major steps of the bx yz pathway are depicted in Scheme 3a
for a general peptide. The bx yz pathway is initiated by
mobilization of the added proton (in Scheme 3a it is located at the
N-terminal amino group; some amino acid side chains might be
more preferred) to the nitrogen of the amide bond to be cleaved.
Nucleophilic attack by the oxygen of the N-terminal neighbor
amide bond on the carbon center of the protonated amide bond
leads to formation of a protonated oxazolone derivative (Yalcin
et al., 1995, 1996; Nold et al., 1997; Paizs et al., 1999; Polce, Ren,
& Wesdemiotis, 2000), whereas the detaching C-terminal
fragment (amino acid or peptide) leaves the parent ion. Under
SCHEME 3.
514
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 3. (Continued )
low-energy collision conditions the loose complex of the
protonated oxazolone derivative and the leaving C-terminal
fragment has a short but finite lifetime, and can undergo a
rearrangement which results in a proton–bound dimer of these
species (Paizs & Suhai, 2002a). Under such circumstances, the
extra proton is shared by the two monomers, and dissociation of
the proton-bound dimer will be determined by the thermochemistry (PA) of the species involved leading to ‘integrated’
formation of bx and yz ions (Paizs & Suhai, 2002a). It is worth
noting here that the neutral counterparts of the yz ions on the
bx yz pathways are oxazolone derivatives. The dissociation
kinetics of the dimer depends on the internal energy distribution
of the ion population, the PA of its monomers, etc. and can be
approximated by using a linear free-energy relationship (Harrison, 1999; Paizs & Suhai, 2002b, 2004):
bx
PAN -term PAC-term
ð1Þ
ln
yz
RTeff
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 neutral
fragments of the corresponding bx yz pathway (i.e., PAs of an
oxazolone derivative and a truncated peptide for the N- and
C-terminal fragments, respectively), and Teff denotes the
‘effective’ temperature.
Most of the experimental results obtained from structural
and energetic studies on small protonated peptides can be
explained based on mechanistic considerations involved in the
bx 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 ion
generated from protonated C6H5CO–Gly–Gly-OH matches the
CID spectra of protonated 2-phenyl-5-oxazolone supporting the
oxazolone structure of the stable b ions.
yz ions have been found by tandem MS to be protonated
truncated peptides or amino acids. The yz ions contain the added
proton as well as a hydrogen that was originally attached to a
515
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PAIZS AND SUHAI
nitrogen N-terminal to the cleaved amide bond and migrated to
the newly formed yz ion (Mueller, Eckersley, & Richter, 1988;
Cordero, Houser, & Wesdemiotis, 1993). This proton migration
step corresponds to the proton transfer reaction between the
oxazolone derivative and the truncated peptide in the protonbound dimer (Scheme 3a) on the bx yz pathway. This proton
transfer step was evaluated (Paizs, Suhai, & Harrison, 2003) for
the peptide H-Gly–Sar–Sar-OH (Scheme 3b) for which y1 ions
can not form on the b2 y1 pathway since the corresponding
oxazolone derivative is a fixed charge and does not have a proton
to be shared with the leaving H-Sar-OH. The breakdown graph of
H-Gly–Sar–Sar-OH is dominated by the b2 ion and y1 appears
only at relatively high energies and originates very probably from
secondary fragmentation of the y2 ion.
The N-terminus has also been probed by using neutral
fragment reionization (NfR) (Nold et al., 1997) experiments,
which give ambiguous results for the neutrals involved. For
protonated C6H5CO–Gly–Phe-OH, the neutral counterpart of
the y1 ion (protonated H-Phe-OH) is 2-phenyl-5-oxazolone
supporting the bx yz pathway. On the other hand, NfR studies on
protonated H-Leu–Gly–Pro-OH have shown that the neutral
counterpart of the y1 ion is a diketopiperazine derivative, making
the general applicability of the bx yz pathway questionable. We
speculate that the unusual behavior of H-Leu–Gly–Pro-OH is
because of the proline effect and other tripeptides (having no Pro
at the C-terminal) fragment on the b2 y1 pathway. This question
is currently under investigation in our laboratory using both
experimental and theoretical tools.
Morgan & Bursey (1994) have reported a linear relationship
between the logarithm of the ratio of y1 and b2 ion abundances
(log(y1/b2)) and the PA of the C-terminal amino acid residue for
protonated tripeptides of the series H-Gly–Gly-Xxx-OH where
Xxx was varied. (Fragmentation of all the H-Gly–Gly-Xxx-OH
tripeptides leads to the same b2 ion, namely, protonated 2aminomethyl-5-oxazolone.) The linearity of the log(y1/b2)
versus PA of Xxx curve can be explained by considering the
corresponding b2 y1 pathway. As mentioned above, the final
step of the bx yz pathway corresponds to the dissociation of the
proton-bound dimer of an oxazolone derivative and a truncated
peptide. The dissociation kinetics can be approximated by using
Equation 1 in which PAN-term is constant (PA of 2-aminomethyl5-oxazolone) whereas PAC-term is varied in the present case
explaining the linearity of the ln(b2/y1) versus PA of Xxx
relationship observed experimentally. Furthermore, the combination of relative ion abundances obtained from the low-energy
MS/MS experiments for the H-Gly–Gly-Xxx-OH series of
tripeptides with appropriate thermochemical data (proton
affinities of Xxx) gives (Paizs & Suhai, 2002b) a reasonable PA
value for 2-aminomethyl-5-oxazolone. (This approach to deriving thermochemical data is similar to Cooks’ kinetic method
(McLuckey, Cameron, & Cooks, 1981).) Similar results have
been obtained for other peptide series like H-Gly-Xxx–Phe-OH
(Harrison, 2002), benzoyl-Gly–Gly-Xxx-OH (Morgan & Bursey, 1995), benzoyl-Xxx-Gly–Gly-OH (Morgan & Bursey,
1995) providing reasonable PA values for intermediates (oxazolone derivatives, F, GG) occurring on various bx yz pathways.
This is possible only if charge-directed cleavage of the
protonated amide bonds of the investigated tripeptides is
dominated by the bx yz pathway.
516
General energetic, kinetic, and entropy factors determining
the activity of the bx yz pathways have recently been
investigated (Paizs & Suhai, 2004). The reactive configurations
of the bx yz pathways (all-trans-amide nitrogen protonated
species) are energetically accessible under low-energy collision
conditions since the corresponding relative energies—calculated
with respect to the most stable structure of the peptide protonated
at the most favored protonation site—are in the range of 15–25
kcal/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 the
existence and facility of proton transfer pathways that connect the
most favored and the amide nitrogen protonation sites at internal
energies well below the threshold energies of the lowest
fragmentation pathways. Once the amide nitrogen protonated
species are reached, concerted formation of the oxazolone ring
and cleavage of the amide bond takes place via a 10–15 kcal/mol
barrier (Paizs et al., 1999; Paizs & Suhai, 2002a) on a time-scale
characteristic of a rearrangement-type reaction. For the peptides
investigated so far, the energy level of the separated final products
is higher than the highest energy transition structure of the
corresponding bx yz pathway. This finding is in line with the
results of metastable ion studies, which indicate small kinetic
energy release (KER) values for the formation of bx and yz ions
of protonated peptides (Polce, Ren, & Wesdemiotis, 2000). In
most of the amide nitrogen protonated species, the oxygen of the
N-terminal neighbor amide bond takes part in charge solvation
(CS) of the—NH2þ—moiety bringing the nucleophilic oxygen
close to the positive carbon center. This ensures that entropy
factors 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 fragmentation
pathways of protonated pentaalanine (Paizs & Suhai, 2004)
permitting for the first time a semi-quantitative understanding of
the IIRs of the MS/MS spectra of a protonated oligopeptide. The
energetics and kinetics of the various bx yz pathways of protonated
pentaalanine have been determined by using theoretical methods
which indicate that at low internal energies, the b4 y1 pathway is
favored compared to b3 y2 and b2 y3. This is in agreement with
the metastable ion and low-energy collision-induced dissociation
mass spectra (Yalcin et al., 1996). At higher energies all the b4 y1,
b3 y2, and b2 y3 PFPs are active behind secondary reactions like
bn ! bn 1 and yn ! yn 1 (for details see below). Equation 1 was
used to approximate the ratio of the bx and yz ions on the particular
bx yz pathways. Applying the necessary proton affinities, such
considerations satisfactorily explain the dominance of the b4 ion
over 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 of
the added proton to form amide nitrogen protonated species.
However, the mechanisms of the cleavages of the aa(2)–aa(3) and
aa(n)–aa(n þ 1) (n > 2) amide bonds differ significantly, therefore, these fragmentation pathways will be described separately.
In general, we refer to the various ‘diketopiperazine’ pathways as
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
diketopiperazine-yN n, where N is the number of amino acid
residues in the peptide and n is used to note the amide bond
(aa(n)–aa(n þ 1)) being cleaved.
The diketopiperazine-yN 2 pathway (Scheme 4) leads to
the formation of diketopiperazine derivatives (Cordero, Houser,
& Wesdemiotis, 1993) as the neutral counterparts of the yN 2
ions. Since diketopiperazine derivatives contain two cis amide
bonds, trans–cis isomerization of the initially trans N-terminal
&
amide bond (aa(1)–aa(2)) is necessary on the diketopiperazineyN 2 pathway (Paizs & Suhai, 2001b). The trans–cis isomerization involves species protonated at the nitrogen of the Nterminal amide bond (aa(1)–aa(2)) (Paizs & Suhai, 2001b). The
next steps involve mobilization of the added proton to the
nitrogen of the aa(2)–aa(3) amide bond (Scheme 4) and attack of
the terminal amino group on the carbon center of the protonated
amide bond. A loose complex of the protonated diketopiperazine
SCHEME 4.
517
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PAIZS AND SUHAI
derivative and the leaving truncated peptide is then formed in
which spontaneous proton transfer to the C-terminal fragment
occurs because the PA of cyclic peptides is much smaller than that
of linear peptides (Nold, Cerda, & Wesdemiotis, 1999). Finally,
the complex dissociates to form the y ion and a neutral
diketopiperazine derivative.
There is no need for cis–trans isomerization of amide bonds
on the diketopiperazine-yN n (n > 2) pathways (Scheme 5) since
cyclic peptides, formed as the neutral counterparts of the yN n
(n > 2) ions, can accommodate all of their amide bonds in the
trans isomerization state (Polce, Ren, & Wesdemiotis, 2000;
Paizs & Suhai, 2004). Therefore, the diketopiperazine-yN n
(n > 2) pathways are initiated by mobilization of the added
proton which is then followed by nucleophilic attack of the N-
terminal amino group on the carbon center of the protonated
amide bond. Formation of the cyclic peptide and cleavage of the
amide bond take place in a concerted manner and yield primarily
the complex of the protonated cyclic peptide and the C-terminal
fragment. Since the proton affinities of cyclic peptides are much
lower that that of linear peptides, the extra proton transfers to the
C-terminal fragment and the complex dissociates to form the
yN n (n > 2) ion and the cyclic peptide as its neutral counterpart.
The size of the cyclic peptide depends on how far the cleaved
amide bond locates from the N-terminus. For yN 3, yN 4, and
yN 5 ions the corresponding neutrals are cyclo-tri-, cylo-tetra,
and cyclo-penta-peptides, respectively.
Many of the experimental results obtained from structural
and energetic studies on small protonated peptides can be
SCHEME 5.
518
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
explained based on mechanistic considerations involved in the
diketopiperazine-yx pathways. yx ions have been found by
tandem MS to be protonated truncated peptides or amino acids.
The yx ions contain the added proton as well as a hydrogen that
was originally attached to a nitrogen N-terminal to the cleaved
amide bond and migrated to the newly formed yx ion (Mueller,
Eckersley, & Richter, 1988; Cordero, Houser, & Wesdemiotis,
1993). This proton migration step corresponds to the proton
transfer reaction between the cyclic peptide and the truncated
peptide in the complex on the diketopiperazine-yx pathways
(Schemes 4 and 5). The N-terminus has also been probed by using
neutral fragment reionization (NfR) (Nold et al., 1997) experiments which have shown that for protonated H-Leu–Gly–ProOH, the neutral counterpart of the y1 ion is a diketopiperazine
derivative.
On the other hand, it seems to be rather difficult to explain
the linear relationship, found between the logarithm of the ratio
of y1 and b2 ion abundances (log(y1/b2)) and the PA of the Cterminal amino acid residue for protonated tripeptides of the
series H-Gly–Gly-Xxx-OH, where Xxx was varied, considering
the diketopiperazine-yN 2 pathway. Wesdemiotis and co-workers (Nold, Cerda, & Wesdemiotis, 1999) have suggested that
protonated oxazolones and cyclic peptides (like diketopiperazine
derivatives) can interconvert in the ion/molecule complex formed
by cleavage of the protonated amide bond in an energetically and
kinetically accessible way (without significant barriers). Therefore, dissociation of the complex will be determined by the
energetics of the b2 and y1 exit channels (PA of the oxazolones
and C-terminal amino acids) explaining the linearity of the
log(y1/b2) versus PA of the Xxx for the series H-Gly–Gly-XxxOH. Paizs & Suhai (2001b) have shown, however, that the
oxazolones and diketopiperazines formed from H-Gly–GlyXxx-OH differ significantly since the isomerization state of the
N-terminal amide bond is cis and trans for these species,
respectively. Since cis–trans isomerization of the amide bond is
time-consuming and requires significant internal energy, it is,
therefore, not likely that the protonated oxazolones and cyclic
peptides can interconvert in the ion/molecule complex formed by
cleavage of the amide bond.
The energetics and the kinetics of the cleavage of the Cterminal amide bond of protonated tripeptides have been
investigated 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 configurations and the transition structures of the b2 y1 pathway (13–24
and 23–29 kcal/mol relative energies, respectively) are more
favored than the corresponding diketopiperazine-yN 2 values
(23–30 and 30–37 kcal/mol relative energies, respectively). On
the other hand, the energy level of the separated final products is
always much deeper for the diketopiperazine-y1 (18–26 kcal/
mol relative energy) than for the b2 y1 (38–40 kcal/mol relative
energy) pathway. If all the pre-dissociation and dissociation
processes of the diketopiperazine-y1 and b2 y1 pathways
occurred under thermodynamic control and the kinetics did not
prevent the formation of intermediates and the dissociation of the
protonated amide bonds, then the CID spectra of protonated HGly–Gly–Gly-OH, H-Gly–Gly–Sar-OH, and H-Gly–Sar–SarOH would be dominated by the y1 ion formed on the
&
diketopiperazine-y1 pathway. While the y1 ion dominates the
CID spectrum of protonated H-Gly–Gly–Sar-OH, fragmentation of protonated H-Gly–Sar–Sar-OH mainly leads to formation of the b2 ion and the y1 ion appears only at higher energies
in the spectrum. This fact can be explained only by assuming that
there is a step on the diketopiperazine-y1 pathway, which is under
kinetic control. In our opinion, this rate-limiting step corresponds
to a trans–cis isomerization of the N-terminal amide bond (Paizs
& Suhai, 2001b).
Recent studies (Paizs & Suhai, 2004) on protonated
oligopeptides indicate the diketopiperazine-yx pathways are
controlled by either energetic or kinetic or entropy factors in the
majority of the cases. The diketopiperazine-yN 2 pathways are
kinetically controlled because trans–cis isomerization of the Nterminal amide bond has to take place prior to the nucleophilic
attack (Paizs & Suhai, 2001b). In the case of the diketopiperazine-yN n (n is ‘average’) pathways trans–cis isomerization of
the N-terminal amide bond is not necessary. The nitrogen of the
terminal amino group can get close to the carbon center of the
protonated amide bond to initiate formation of the cyclic peptide.
However, small cyclic peptides accommodating only trans amide
bonds suffer from significant ring strain leading to energetically
disfavored fragmentation products. As the size of the cyclic
peptide increases (cleavage far from the N-terminus), the cyclic
peptides will suffer from less and less ring strain leading to
energetically more favored diketopiperazine-yN n (n is ‘large’)
pathways. However, these diketopiperazine-yN n pathways will
be discriminated by entropy effects. This is because of the fact
that the amide nitrogen protonated species are effectively
solvated by nearby amide oxygens and the terminal amino group
must compete with this kind of CS to get close to the center of the
protonated amide bond. While CS of the—NH2þ—moiety by the
terminal amino group is energetically feasible, the number of
such species will be small compared to the large number of
such species where amide oxygens provide stabilization. This
means that dissociation of protonated oligopeptides on the
diketopiperazine-yN n pathways if the amide bond to be cleaved
is located far from the N-terminus is disfavored because of
entropy factors.
3. Amide Oxygen Peptide Fragmentation Pathways
In the previous sections, it was repeatedly shown that amide
nitrogen protonated species are responsible for cleavage of the
amide bonds. This is because of the fact that protonation at the
nitrogen weakens the amide bond and makes the carbon center of
the amide bond more positive and therefore a possible target of
nucleophilic attack. Pathways like bx yz and diketopiperazineyx involve amide nitrogen protonated species as reactive
configurations. Modeling the energetic and kinetic details of
the bx yz pathways of protonated pentaalanine led to a semiquantitative understanding of the MS/MS spectra of oligopeptides. Contrary to the success of mechanisms that involve amide
nitrogen protonated species, other pathways based on protonation at amide oxygens regularly appear in the literature. In the
following, we shortly describe the ‘‘amide oxygen’’ pathways
and summarize why it is unlikely that these dissociation channels
are active under low-energy collision conditions.
519
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PAIZS AND SUHAI
It is well known that protonation at amide oxygens is
energetically favored compared with protonation at amide
nitrogens. Based on this fact a mechanism (Arnot et al., 1994;
Reid, Simpson, & O’Hair, 1998; Vaisar & Urban, 1998; Wysocki
et al., 2000) was proposed for the formation of b and y ions which
does not involve any amide nitrogen protonated species. The
mechanism applied to a general peptide is briefly outlined in
Scheme 6. The amide oxygen pathways are initiated by
mobilization of the added proton to reach amide oxygens. This
step requires less energy than mobilization of the added proton to
amide nitrogens. In the next steps, a five-membered ring is
formed by attack of the N-terminal neighbor amide oxygen on the
carbon center of the carbonyl-O-protonated amide group, and
then an 1,1-elimination occurs resulting in loss of the C-terminal
fragment and formation of a protonated oxazolone. The N- and Cterminal fragments can separate with or without proton transfer
leading to b and y ions.
Theoretical investigation of the PES of a large number of
protonated peptides (H-Gly–Gly–Gly-OH, H-Gly–Gly–ProOH, 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 species
containing the five-membered ring exist but are energetically not
favored (Paizs & Suhai, 2001b). Actually, the relative energy of
such species is often higher than even those of the most stable
amide nitrogen protonated species of the same compounds. The
next step on the amide oxygen pathway corresponds to cleavage
of the amide bond by 1,1 elimination on the Camide carbon of the
already high-energy amide oxygen protonated five-membered
ring-containing species (Scheme 6). The corresponding transition structure involves a highly strained four-membered ring
leading to a large barrier (Paizs et al., 2001). For larger peptides
this barrier could be reduced by catalysis of the proton transfer
step involving other basic sites of the peptide. It seems likely,
SCHEME 6.
520
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
however, that even after reducing the barrier, the corresponding
transition structures will be less favored than TSs on the bx yz
pathways. These observations indicate that the amide oxygen
pathways do not contribute significantly to the formation of
sequence ions of protonated peptides.
B. Dissociation of the N-terminal (aa(1)–aa(2)) Amide
Bond of Underivatized Protonated Peptides
Charge-directed dissociation of the underivatized N-terminal
amide bond of protonated peptides significantly differs from the
cleavage 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 bond
cleavage or by rearrangement-type reactions. Most of the
fragmentation reactions of protonated peptides excited under
low-energy collision conditions belong to the latter class,
because even the excited ions do not have enough energy to
fragment on direct bond cleavage pathways which are facile only
if significant energy is deposited into the ions. It is not surprising
that low-energy pathways which include direct bond cleavage of
the protonated amide bond are active mainly for the N-terminal
amide bond (a1 yx pathway, Paizs & Suhai, 2001a) for which
the only available backbone nucleophile that can induce
fragmentation is the N-terminal amino group. The corresponding
rearrangement-type reaction (aziridinone pathway, Cordero,
Houser, & Wesdemiotis, 1993) however, includes the formation
of an energetically disfavored three-membered ring giving rise to
the possibility of the a1 yx pathway to compete efficiently for
mainly dipeptides and small peptides containing amino acid
residues with high proton affinities at the N-terminus. In our
opinion, the majority of ions originating from dissociation of the
N-terminal (aa(1)–aa(2)) amide bond of underivatized protonated peptides are formed on the a1 yx pathways. It is worth
noting here that one can easily shut down the a1 yx and
aziridinone pathways by, for example, introducing through
acetylation of the N-terminus a good nucleophile (amide oxygen)
N-terminal to the aa(1)–aa(2) amide bond that can already initiate
rearrangement-type cleavage of the amide bond.
1. a1 yx Peptide Fragmentation Pathways
The major steps of the a1 yx pathway are illustrated in
Scheme 7. Being a charge-directed dissociation channel, the
a1 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 protonated
imine (N-terminal fragment), truncated peptide, or amino acid
(C-terminal fragment) and CO. After loss of the weakly bonded
CO, a proton-bound dimer of the N- and C-terminal fragments is
formed. Under low-energy conditions the lifetime of this dimer is
long enough so that numerous proton transfers can take place
between the two fragments in the dimer. Therefore, there are two
exit channels through which the proton-bound dimer can dissociate without passing a barrier in the next step to form either a1
or yx ions. The dissociation kinetics of the dimer depends on the
internal energy distribution of the ion population, the PA of its
monomers, etc. and can be approximated by using a linear free-
&
energy relationship (Harrison, 1999; Paizs & Suhai, 2002b; Paizs
et al., 2004):
a1
PAN -term PAC-term
ln
yx
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 neutral
fragments of the corresponding a1 yx pathway (that is, PAs of
an imine and a truncated peptide for the N- and C-terminal
fragments, respectively), and Teff denotes the ‘effective’
temperature.
The major characteristics of the a1 yx pathway can be used
to explain most of the corresponding experimental results
obtained for protonated dipeptides and small peptides. yx ions
have been found by tandem MS to be protonated truncated
peptides or amino acids. The yx ions contain the added proton as
well as an H-atom that was originally attached to the N-terminal
nitrogen and migrated to the newly formed yx ion (Mueller,
Eckersley, & Richter, 1988; Cordero, Houser, & Wesdemiotis,
1993). This latter step corresponds to the proton transfer reaction
between the N- and C-terminal fragments in the proton-bound
dimer (Scheme 7) on the a1 yx pathway. On the other hand,
when the C-terminus is eliminated as a neutral fragment, neutral
fragment reionization (NfR) experiments have shown it to be
cleaved as an intact amino acid (Cordero, Houser, & Wesdemiotis, 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 ions
characteristic to the MeCH=NH imine produced as the neutral
counterpart of the y1 ion on the a1 yx pathway. The uncertainty
regarding the interpretation of the NfR spectrum could no doubt
be resolved if the corresponding experiments be repeated for a
system where the y1 is the major fragmentation product.
Harrison et al. (2000) have investigated the major lowenergy fragmentation pathways of many protonated dipeptides
paying special attention to the various structural factors affecting
the a1/y1 abundance ratio. They have found that for a series of
protonated dipeptides H-Val-Xxx-OH, ln(a1/y1) is a linear
function 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 the
linearity of the plot, only the slope is changed.) As mentioned
above, the final step in the a1 yx pathway corresponds to the
dissociation of the proton-bound dimer of an imine and an amino
acid for dipeptides (HN=CHCH(CH3)2 and Xxx, the varied
amino acid for the H-Val-Xxx-OH series of peptides, respectively). The dissociation kinetics can be approximated by using
Equation 2 in which PAN-term is constant whereas PAC-term is
varied in the present case explaining the linearity of the ln(a1/y1)
versus PA of Xxx relationship observed experimentally. Furthermore, the combination of relative ion abundances obtained from
metastable ion fragmentation for the H-Val-Xxx-OH series of
dipeptides with appropriate thermochemical data (proton
affinities of Xxx) gives (Paizs et al., 2004) a reasonable PA value
for HN=CHCH(CH3)2 (imine derived from Val). (This approach
to derive thermochemical data is similar to Cooks’ kinetic
method (McLuckey, Cameron, & Cooks, 1981).) This is possible
521
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PAIZS AND SUHAI
SCHEME 7.
only if charge-directed cleavage of the amide bond of the
investigated dipeptides is dominated by the a1 yx pathway.
Harrison et al. (2000) have investigated the fragmentation of the
H-Xxx-Phe-OH dipeptide series for which ln(a1/y1) gave poor
correlation with the PA of H-Xxx-OH. We have recently shown
(Paizs et al., 2004) that reasonable correlation can be obtained if
one considers the ln(a1/y1) versus PA of the imines derived from
Xxx relationship for the H-Xxx-Phe-OH data, in accordance with
the general characteristics of the a1 yx pathway. It is worth
noting here that Equation 2 can be used only for the low-energy
522
fragmentation regime since the proton equilibration implicit in
the corresponding mechanism may not be established if the life
time of the proton bound dimmer is too short.
The energetics of the fragmentation of protonated dipeptides
has been investigated by using energy-resolved CID-MS
(Klassen & Kebarle, 1997) and SID-MS (Laskin, Denisov, &
Futrell, 2000). Klassen & Kebarle (1997) have determined the
kinetic shift corrected appearance energy of the a1 ion of protonated H-Gly–Gly-OH to be 43.7 kcal/mol. Laskin, Denisov, &
Futrell (2000) have reported critical energies for the H-Ala–Ala-
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
OHHþ ! H-Ala–Ala-OHHþ–CO, H-Ala–Ala-OHHþ ! a1,
and H-Ala–Ala-OHHþ ! y1 reactions at 44.3, 48.7, and
47.5 kcal/mol, respectively. These data are in keeping with
computational results on the a1 yx pathway (Paizs & Suhai,
2001a) showing that loss of CO is energetically more favored
than formation of a1 or y1 for protonated H-Gly–Gly-OH. On the
other hand, the theoretical and experimental data for formation of
a1 and y1 ions cannot be directly compared since the reaction
pattern used to integrate the kinetics (Laskin, Denisov, & Futrell,
2000) of the various dissociation channels did not include the
a1 yx pathway and relied on direct formation of a1 and y1 from
the parent ion. This suggests that caution must be observed in
deriving energetic parameters from energy resolved MS studies,
if the underlying reaction mechanisms are not fully known.
Theoretical investigation of the dissociation of the amide
bond of a few dipeptides has indicated (Paizs & Suhai, 2001a;
Paizs et al., 2004) that the transition structure belonging to
concerted cleavage of the amide and the Ca-Camide (CHR1-CO
for Scheme 7) bonds lies at 32–38 kcal/mol relative energy and
increases 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 and
the fully separated final products (either a1, C-terminal amino
acid and CO or y1, imine derived from the N-terminal amino acid
and CO) have relative energies at 9–15 and 35–45 kcal/mol,
respectively. This energetics is in keeping with the results of
metastable ion studies which indicate small KER values for
the 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 also
used to explain the energy-dependence of the mass spectra of
protonated dipeptides. For example, unimolecular decomposition 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% under
low-energy CID, from which it is evident that loss of CO and the
formation 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 loss
disappears. At very low energies many fragmenting species
which had enough energy to get through the a1 yx transition
structure do not fully dissociate from the proton-bound dimer
phase (CO loss peak) since this step requires significant energy.
The fragmenting proton-bound dimers, which have relatively low
internal energies choose the energetically favored way leading to
y1 formation. As the internal energy increases one sees more y1
and a1 ions and less abundant CO loss. Finally, at high energies
there is no time for proton-equilibration in the proton-bound
dimer, 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 in
Scheme 8. Being a charge-directed dissociation channel, the
aziridinone pathway is initiated by mobilization of the added
proton, which has to reach the nitrogen of the N-terminal amide
bond. The aziridinone transition structure corresponds to
concerted cleavage of the protonated amide bond and formation
&
of the aziridinone ring. Under low-energy collision conditions
the N- and C-terminal fragments (an aziridinone derivative and a
truncated peptide, respectively) do not separate immediately and
form a proton-bound dimer (Harrison et al., 2000) in which
numerous proton transfers can occur between the two monomers.
(The nitrogen protonated aziridinone derivative is stable in the
proton-bound dimer (Harrison et al., 2000).) Dissociation of the
proton-bound dimer leads to yx ions if the C-terminal fragment
keeps the added proton during spatial separation of the
fragments. On the other hand, the nitrogen protonated aziridinone derivative is not stable (Harrison et al., 2000) and decomposes to a1 and CO if the extra proton is kept by the N-terminal
fragment. (The O-protonated form of aziridinone is stable but the
N-protonated isomer is formed in the aziridinone pathway.)
Mechanistic considerations involved in the aziridinone
pathway can explain some of the experimental results obtained
for protonated dipeptides and small peptides. yx ions have been
found by tandem MS to be protonated truncated peptides or
amino acids which contain the added proton as well as an H-atom
that was originally attached to the N-terminal nitrogen and
migrated to the newly formed yx ion (Mueller, Eckersley, &
Richter, 1988; Cordero, Houser, & Wesdemiotis, 1993). Migration of the proton on the aziridinone pathway corresponds to the
proton transfer reaction between the N- and C-terminal fragments in the proton-bound dimer of the aziridinone derivative and
the truncated peptide (Scheme 8) and involves one of the
hydrogens originally attached to the terminal amino group. The
aziridinone pathway is in line also with the results of NfR
experiments which show that if the C-terminus is eliminated as a
neutral fragment then it is cleaved as an intact amino acid or small
peptide (Cordero, Houser, & Wesdemiotis, 1993). NfR experiments on protonated H-Ala–Ala-OH gave some support to the
aziridinone pathway since the NfR spectra contained lowabundance peaks that are signature ions of the corresponding
aziridinone derivative.
The aziridinone pathway could explain also the linearity of
the log(a1/y1) versus PA of Xxx curves for the series of H-ValXxx-OH dipeptides (Harrison et al., 2000) by considering that
dissociation of the proton-bound dimer of the aziridinone
derivative and the C-terminal fragment is determined by the
proton affinities of the corresponding neutrals. Unfortunately,
this hypothesis cannot be quantitatively assessed as proton
affinities are available neither experimentally nor computationally for aziridinone derivatives since the N-protonated form is not
stable. On the other hand, combination of relative ion abundances
obtained from low-energy MS/MS experiments for the H-ValXxx-OH series of dipeptides with appropriate thermochemical
data (proton affinities of Xxx) gives (Paizs et al., 2004) a
reasonable PA value for the intermediate (HN=CHCH(CH3)2) on
the corresponding a1 yx pathway. Since it is not likely that the
PAs of HN=CHCH(CH3)2 and the corresponding aziridinone
derivative in the proton-bound dimer of the aziridinone pathway
are equal, this fact strongly suggests that only a small fraction of
the a1 and y1 ions are formed on the aziridinone pathway.
Theoretical investigation of the dissociation of the amide
bond of a few dipeptides indicated (Paizs & Suhai, 2001a; Paizs
et al., 2004) that the aziridinone pathway is both energetically and
kinetically less favored than the corresponding a1 yx pathways.
The aziridinone transition structures lie at 44–50 kcal/mol
523
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PAIZS AND SUHAI
SCHEME 8.
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 calculations suggest that the corresponding unimolecular rate constants
are much smaller than the corresponding a1 yx values. Furthermore, the separated final products are energetically disfavored on
the aziridinone pathway compared to a1 yx if yx ions are
formed. (The three-membered aziridinone ring is energetically
more demanding than separated CO and the corresponding
imine.)
There seems to be a controversy between the mechanistic
considerations involved in the aziridinone pathway and the
tendency to form [MH CO]þ ions for some protonated
dipeptides. Formation of a1 ions on the aziridinone pathway is
proposed via separation of the proton-bound dimer to form the
nitrogen protonated aziridinone derivative, which is stable only
in the complex and decomposes as the C-terminal fragment
repels out. It is not likely that [MH CO]þ ions are formed after
the dissociation of the protonated aziridinone by reunion of the
protonated imine (a1) and the C-terminal fragment. Also, it seems
to be rather difficult to explain the energy dependence of the MS/
524
MS spectra of dipeptides like H-Gly–Gly-OH based on the
aziridinone pathway.
C. Charge Directed Amide Bond Cleavage via Attack
of Nucleophilic Groups of Amino Acid Side Chains
Beside the PFPs involving interaction of only backbone
functional groups, some peptides can dissociate via amide bond
cleavage initiated by specific amino acid side chains (Scheme 9).
The most important such reactions involve nucleophilic attack by
the histidine, glutamine, asparagine, lysine, and arginine side
chains on the C-terminal adjacent nitrogen protonated amide
bond and lead to various cyclic (non-oxazolone derivative) Cterminal fragments. Since the reactive configurations of the
corresponding side chain initiated and bx yz pathways
(Scheme 9) are species containing the nitrogen protonated amide
bond, competition of the two dissociation channels can lead to
mixture of the two isomeric forms of the N-terminal fragments.
While the charge directed dissociation of amide bonds is
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 9.
dominated in most of the cases by the general bx yz pathways,
side chain induced cleavage C-terminal to histidine and
glutamine 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 instead
of backbone nucleophiles like the amide oxygens or the nitrogen
of the terminal amino group. The histidine effect has been studied
by Wysocki et al. (2000) and Farrugia, Taverner, & O’Hair (2001)
in detail.
Cleavage at the C-terminal side of histidine is preferred for
many peptides if the number of added protons is larger than the
number of Arg residues present (Wysocki et al., 2000). Because
of its high PA the His side chain is very probably protonated in the
lowest energy structures of such peptides (Scheme 10). The first
step of the reaction mechanism involves mobilization of the
proton located at the His side chain to the nitrogen of the Cterminal neighbor amide bond, which is followed by nucleophilic
attack of the imidazole nitrogen on the carbon of the protonated
amide 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 to
allow proton transfer between the monomers and formation of
both bx and yz ions.
The histidine effect has been systematically probed
(Wysocki et al., 2000) by MSn experiments, varying the adjacent
amino acid residues in the peptide, alkylation of the His side
chain, etc. For example, the MS/MS spectrum of doubly
protonated H-Arg–Val–Tyr–Ile–His–Pro–Phe-OH is dominated by the b5þ and y2þ ion pair suggesting that the proton
transfer step of Scheme 10 is possible. Substituting Pro by Ala
results in dominance of b52þ and b62þ. This can be explained by the
difference between the PAs of H-Pro–Phe–OH and H-Ala–PheOH. Finally, alkylation of the His side chain shuts down the
proton transfer part of the mechanism depicted in Scheme 10
leading to dominance of bx2þ ions in the MS/MS spectra. Also,
ab initio calculations indicate that the non-classical bx ion of
Scheme 10 is stable (Farrugia, Taverner, & O’Hair, 2001).
2. Peptide Fragmentation Pathways Involving the
Amide Oxygen of the Gln and Asn Side Chains
The amide moieties of the Gln and Asn side chains can be
involved in reactions leading to cleavage of the amide bond Cterminal to these amino acid residues. The corresponding PFPs
525
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PAIZS AND SUHAI
SCHEME 10.
are initiated by mobilizing the added proton which should reach
the nitrogen of the amide bond to be cleaved (Schemes 9 and 11).
Nucleophilic attack by the amide oxygen of the Gln or Asn side
chain on the carbon center of the protonated amide bond leads to
formation of cyclic isoimide (Jonsson et al., 2001a,b; Farrugia,
O’Hair, & Reid, 2001; Harrison, 2003) derivatives, whereas the
detaching C-terminal fragment (amino acid or peptide) leaves
the parent ion. The same amide bond can also be cleaved on the
corresponding bx yz pathway which leads to oxazolone
formation and therefore raises the possibility of forming isomers
of the N-terminal fragment (Scheme 9). Under low-energy
collision conditions the loose complex of the protonated isoimide
derivative and the leaving C-terminal fragment has a short but
finite lifetime allowing rearrangement of and proton transfers
between the fragments. Therefore, dissociation of the dimer will
526
be determined by the thermochemistry (PA) of the species
involved leading to ‘integrated’ formation of bx and yz ions
similarly to the bx yz pathways.
Farrugia, O’Hair, & Reid (2001) have investigated b2 ions
derived from protonated N-acyl Gln and Asn methyl esters using
multistage MS and ab initio techniques. These b2 ions fragment
by losing both CO (for more details, see the chapter on the
bx ! ax pathway) and CH2CO, indicating that both the oxazolone
and the isoimide forms are present in the mass spectrometer. Ab
initio calculations have indicated that the oxazolone form is more
stable than the isoimide isomer for both the Gln and Asn
containing b2 ions. However, the difference between the relative
energies of the two forms is much smaller for the former
(2 kcal/mol) than for the latter (11 kcal/mol). Cleavage of the
amide bond C-terminal to Asn and Gln involving nucleophilic
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 11.
attack of the side chain amide nitrogen has also been investigated
by determining the relative energies of the corresponding isomers
of the b2 ions. These calculations have indicated that such a
reaction is energetically disfavored.
Harrison (2003) has investigated the fragmentation pathways of protonated H-Gly–Gln–Gly-OH using multistage MS.
The b2 ion shows a variety of fragmentation reactions including
loss of CO (for more details, see the chapter on the bx ! ax
pathway) and a glycine residue. The former reaction is
characteristic of b ions with the classical oxazolone structure
whereas the latter dissociation channel can be derived only from
the isoimide isomer. Jonsson et al. (2001a,b) have investigated
the fragmentation reactions of tryptic and synthetic peptides
containing the Gln–Gly sequence motif. These authors found
that facile Gln–Gly cleavage occurs when an Xxx-Gln–Gly-Yyy
sequence is present in the peptide, where Xxx is any amino acid
and Yyy is any amino acid except for Gly. Also, a model peptide
containing Asn instead of Gln showed less dominant dissociation
of the Xxx-Gly amide bond in line with the theoretical data
obtained by Farrugia, O’Hair, & Reid (2001) on the stability of
the oxazolone and isoimide forms of the b2 ions derived from the
N-acyl Asn and Gln methyl esters.
527
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PAIZS AND SUHAI
3. Peptide Fragmentation Pathways Involving
the Lys and Arg Side Chains
Peptides containing only Lys as a basic amino acid are most
probably protonated at the e-amino group of the Lys side chain.
Mobilization of the added proton can lead to various amide
nitrogen protonated species resulting in formation of bx and yz
ions on the bx yz pathways. If the extra proton reaches the LysXxx amide bond, the side chain of Lys can also initiate amide
bond cleavage to form a caprolactam derivative (Scheme 12).
Yalcin & Harrison (1996) have investigated the fragmentation
pathways of protonated H-Lys-Gly-OH and other Lys derivatives. The nominal acylium ion at m/z ¼ 129 is produced in both
metastable ion and collision induced fragmentation of the
investigated Lys derivatives. The product ion spectra of ion
m/z ¼ 129 is very similar to that of protonated a-amino-ecaprolactam confirming the dissociation mechanism of Scheme
12. The PFPs of protonated H-Lys-Gly-OH have also been
studied by using quantum chemical and RRKM calculations
(Csonka et al., 2001) further confirming the assumption of
caprolactam formation.
Farrugia, O’Hair, & Reid (2001) have investigated the b2 ion
derived from protonated N-acyl Lys methyl ester using multistage 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 is
present in the mass spectrometer. Ab initio calculations have
suggested that the oxazolone form (Scheme 9) is less stable than
the caprolactam isomer by 2 kcal/mol.
Kish & Wesdemiotis (2003) have investigated the PFPs of
protonated H-Gly–Gly–Lys–Ala–Ala-OH utilizing multistage
MS in an ion trap instrument. Formation of the corresponding b3
ion is more significant than that of the b3 ion of protonated HTyr–Gly–Gly–Phe–Leu-OH. The b3 ion derived from H-Gly–
Gly–Lys–Ala–Ala-OH fragments by losing H2O and Gly–Gly
with 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 the
caprolactam (Scheme 13) structure of the b3 ion.
The b2 ion derived from protonated N-acyl Arg methyl ester
has recently been investigated using multistage MS and ab initio
techniques (Farrugia, O’Hair, & Reid, 2001). The MS/MS/MS
spectrum does not show CO loss indicating a non-classical
structure of the b2 ion. This behavior is in line with the
mechanism depicted in Scheme 13 which involves mobilization
of the extra proton originally sequestered by the Arg side chain to
the Arg-Xxx amide nitrogen and subsequent nuclephilic attack
by the neutral guanidino group to induce amide bond cleavage
SCHEME 12.
528
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 13.
and formation of a six-membered ring. Ab initio calculations
(Farrugia, O’Hair, & Reid, 2001) have suggested that the
oxazolone form (Scheme 9) is less stable than the nonclassical b2 isomer by 2 kcal/mol. The MS/MS spectrum of
protonated H-Arg–Asp-NH2 shows an abundant peak at
m/z ¼ 157 (Paizs et al., 2002) representing the usually unstable
b1 ion, whose formation can be explained by the mechanism of
Scheme 13.
It is worth noting here that Farrugia & O’Hair (2002) have
recently discovered an interesting gas phase rearrangement for
protonated arginine-containing dipeptides. For protonated HArg–Gly-OH and H-Gly–Arg-OH, the rearrangement leads to
identical MS/MS spectra. DFT calculations and MS/MS/MS
experiments suggest a mechanism, which involves formation of a
common cyclic intermediate. Recent MS/MS results on protonated H-Arg–Gly–Gly-OH and H-Gly–Gly–Arg-OH indicate
that this rearrangement is characteristic of dipeptides, and that
larger Arg-containing peptides fragment by involving other PFPs
(Paizs & Somogyi, unpublished results).
D. Peptide Fragmentation Pathways Leading
to Loss of Small Neutrals
The MS/MS spectra of protonated peptides often contains
fragment ions originating from losses of small neutrals like
water 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 are
formed on charge-directed PFPs. There are three possibilities for
the water loss (Ballard & Gaskell, 1993) of protonated peptides
involving dehydration of the C-terminal, Asp, and Glu COOH
group, at backbone amide oxygens, and at side chains of Ser and
Thr. For some peptides only one of the corresponding pathways
dominates whereas the fragmentation of other peptides show
mixing of the above channels. Loss of ammonia occurs from the
side chains of Arg, Lys, Asn, and Gln. These reactions are
practically important since tryptic peptides contain either Arg or
Lys at the C-terminus leading to usually abundant yz-NH3 series.
It is worth noting here that elimination of CO can formally be
529
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PAIZS AND SUHAI
considered as a PFP leading to loss of a small neutral. There are
two practically important cases involving loss of CO from bx ions
and 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 by
mobilization of the extra proton, which has to reach the hydroxyl
group. 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 the C-terminus, proton transfer to
the OH group, breaking of the HO–C bond, and formation of an
oxazolone ring occur concertedly but asynchronously. The first
two events occur at the early stage of the fragmentation whereas
oxazolone formation takes place only at the final stage of the
reaction. Multistage MS and H/D exchange experiments by Reid,
Simpson, & O’Hair (1999) proved the expected oxazolone
structure 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.
530
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
These investigations indicated that protonated H-Thr–Arg–
Lys–Arg-OH dehydrates nearly exclusively at the C-terminal
COOH group. Water loss from the Asp and Glu side chains occurs
via a similar mechanism shown in Scheme 14 (route 1) and the
main difference between the reactions of the backbone and side
chain COOH groups is in the structure of the resulting cyclic
products. A practically significant side chain activity occurs if
peptides and/or their yz ions contain Glu at the N-terminal
position. For such cases, formation of pyroglutamic acid
(Scheme 14, route 2) is usually facile and accompanied by
abundant loss of water.
The MS/MS spectra of protonated [18O2] H-Arg–Pro–Pro–
Gly–Phe-OH (Ballard & Gaskell, 1993) show that dehydration
occurs primarily through the loss of H216O involving one or more
of the backbone amide oxygens. MS/MS/MS studies indicated
that the primary site of water loss involves the carbonyl oxygen of
the amide bond nearest to the N-terminus. Reid, Simpson, &
O’Hair (1999) have proved that the b4 and b5 ions derived from
protonated H-Gly–Gly–Gly–Gly-OH and H-Gly–Gly–Gly–
Gly–Gly-OH do not have oxazolone structure which is expected
if dehydration involves the C-terminal COOH group. Also, the
MS/MS spectrum of methylated H-Gly–Gly–Gly–Gly-OH
suggests that elimination of water occurs involving an amide
oxygen. While some efforts to elucidate the mechanism behind
the backbone water loss are described (Reid, Simpson, & O’Hair,
1999), atomistic details of the underlying chemistry are not
known yet.
Dehydration involving the Ser and Thr side chains has been
studied by Ballard & Gaskell (1993) and Reid, Simpson, &
&
O’Hair (2000). The corresponding reaction (Scheme 15) is
initiated by mobilization of the extra proton to the Ser or Thr side
chain oxygen which is followed by nucleophilic attack of the
C-terminal adjacent amide oxygen to form a stable five- or sixmembered ring, respectively. Protonated tris-methyl ester
derivative of the delta-sleep-inducing peptide fragments via loss
of water despite its lack of free carboxylic acid groups (Ballard &
Gaskell, 1993). Analysis of the MS/MS/MS spectrum and
protecting the Ser OH group suggest that water is eliminated
from the serine side chain for this peptide.
2. Ammonia-Loss Peptide Fragmentation Pathways
Charge directed loss of ammonia occurs from the side chains of
Asn, Gln, Lys, and Arg amino acid residues. (No data are reported
on ammonia loss involving the N-terminal amino group in the
literature.) A common characteristic of the ammonia loss PFPs is
that protonation at the corresponding side chain is required. For
those cases when NH3 is eliminated from the Lys and Arg
side chains, mobilization of the extra proton is not required since
the corresponding e-amino and guanidino groups are usually the
most favored protonation sites of peptides. For deamidation from
the Asn and Gln side chains, mobilization of the extra proton
from more favored protonation sites like the N-terminal amino
group of basic amino acid side chains is necessary.
Loss of ammonia occurs from Lys-containing peptides via
SN2-type reactions (Csonka et al., 2001) which lead to
elimination of the nitrogen of the e-amino group. (15N-labeling
experiments (Dookeran, Yalcin, & Harrison, 1996) have shown
SCHEME 15.
531
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PAIZS AND SUHAI
that elimination of ammonia specifically involves the nitrogen of
the side chain of protonated H-Lys-OH.) If Lys is located at the Nterminus of the peptide under investigation, the most likely PFP
(Csonka et al., 2001) involves the nucleophilic attack by the Nterminal amino group (Scheme 16, reaction 1) leading to
formation of pipecolic acid at the N-terminus. It is worth noting
here that nucleophilic attack by the Lys-Xxx amide oxygen to
induce NH3 loss from the Lys side chain is also possible but this
pathway is kinetically disfavored with respect to the PFP
involving the a-amino group.
If Lys is located far from the N-terminus, entropy factors
preclude elimination of ammonia via nucleophilic attack by the
N-terminal amino group. In such circumstances, ammonia loss is
initiated by the Lys-Xxx amide oxygen or the C-terminal COOH
group (Scheme 16, reaction 2). This PFP is of practical
importance when tryptic peptides with Lys at the C-terminus
are dissociated.
Elimination of NH3 from the protonated guanidino group of
the Arg side chain has recently been investigated by using
theoretical methods (Csonka, Paizs, & Suhai, 2004, Modeling of
the gas-phase ion chemistry of protonated arginine J Mass
Spectrom, accepted for publication). These calculations indicate
that beside the usual NeH–Cþ(NhH2)(Nh0 H2) form of the
protonated guanidino group other tautomers like –NeH–
C(NhH3þ)(Nh0 H) or –Ne–C(NhH3þ)(Nh0 H2) (Scheme 17) involving the preformed—NH3 group can exist in mass spectrometers. Loss of ammonia from the Arg side chain occurs very
probably by SN1 substitution on the central z-carbon of the
guanidine group from the –NeH–C(NhH3þ)(Nh0 H) or –Ne–
C(NhH3þ)(Nh0 H2) tautomers. The resulting protonated carbodiimide group (Scheme 17) can be stabilized by intramolecular
attack of nearby nucleophiles leading to ring formation.
Theoretical studies on protonated H-Arg-OH indicate this
pathway requires at least 45 kcal/mol internal energy. It is
worth noting here that BIRD of protonated bradykinin (Price,
Schnier, & Williams, 1996) leads to elimination of NH3 with 1.3
eVactivation energy. The theoretical value for protonated H-ArgOH and the BIRD data for protonated bradykinin differ
significantly, suggesting that either SB structure of protonated
bradykinin (Schnier et al., 1996) influences the energetics of the
PFP depicted in Scheme 17 or an alternative lower-energy route
exists for the elimination of NH3 from the Arg side chain.
Loss of ammonia from the Asn and Gln residues is initiated
by mobilization of the extra proton to the nitrogen of the side
chain amide bond, the carbon center of which becomes a likely
target of nucleophilic attack. If Gln is located at the N-terminus,
SCHEME 16.
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FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 17.
the a-amino group can be involved as attacking nucleophile
leading to facile pyroglutamate formation (Scheme 18, reaction
1). If Gln and Asn are located far from the N-terminus, amide
oxygens can initiate the deamidation (Scheme 18, reaction 2).
The corresponding chemistry is complicated and involves
formation of rather unexpected products (Harrison, 2003). For
example, elimination of ammonia from protonated H-Gly–GlnGly-OH and H-Gly–Gly–Gln-OH (Scheme 18, reaction 3) leads
at least partially to formation of protonated H-Gly–Gln–Pyr-OH
and H-Gly–Gly–Pyr-OH, respectively, in a reaction with a yet
unknown mechanism.
E. Fragmentation Pathways of bx and yz Ions
The MS/MS spectra of protonated peptides frequently show
whole or partial series of bx and yz ions. As discussed above, the
primary source of these ions is the peptide parent ion. However,
bx and yz ions can also be formed from higher b and y ions
533
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PAIZS AND SUHAI
SCHEME 18.
(Ballard & Gaskell, 1991; Yalcin et al., 1996). The yz ions are
truncated peptides, their fragmentation behavior can be
described 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 to
specific fragmentation channels like elimination of CO (bx ! ax
534
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 relatively
weak bx ion signals when acquired in triple quadrupole or
quadrupole time-of-flight (Q-TOF) mass spectrometers. This
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
behavior is very probably because of facile bx ! bx 1 pathways
which 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 Cterminal Lys or Arg side chains.
It is also worth noting here that formation of internal
fragments 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 the
Harrison and Glish groups (Yalcin et al., 1996; Vachet, Ray, &
Glish, 1998). Harrison and co-workers have investigated the
reactions of higher bx ions of protonated H-Gly–Gly–Gly–GlyOH, 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 in
reactions occurring with relatively low KERs on the metastable
ion time scale. Based on the observed fragmentation characteristics, it was proposed that the investigated bx ions have the
protonated oxazolone structure. This suggestion has recently
been confirmed in a theoretical study on the bx ions of protonated
pentaalanine (Paizs & Suhai, 2004).
However, the atomistic details of the underlying reaction
mechanism are not yet clarified. Harrison suggested (Yalcin et al.,
1996) a one-step mechanism in which the C-terminal adjacent
amide oxygen attacks the –C=Nþ– carbon of the oxazolone
ring and induces elimination of an aziridinone derivative
(Scheme 19). Fang et al. (1999) have studied this mechanism
utilizing theoretical methods and determined the barrier to the
corresponding transition structure at 77 kcal/mol which is
clearly too high for mass spectrometers operating under lowenergy collision conditions. These authors have also investigated
the bx ! ax ! bx 1 indirect pathway (Scheme 19, route 2) which
requires at least 40 kcal/mol internal energy and seems to be
more 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 release
of kinetic energy (T1/2 ¼ 0.3–0.5 eV) indicating that the indirect
bx ! ax ! bx 1 pathway cannot be responsible for the formation
of 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 transform
and double resonance techniques in conjunction with a quadrupole ion trap. By ejecting some product ions as they are formed,
further dissociation of these ions can be systematically
investigated in this way. Vachet, Ray, & Glish (1998) showed
that 50 % of the b3 ions of various Leu-enkephaline derivatives
originated directly from the corresponding b4 ions under the
moderately energetic conditions applied. These experimental
data 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 separated
by the characteristic 28 Da mass difference which facilitates
&
identification of members of the b ion series in the MS/MS
spectra of protonated peptides. Also, under low-energy collision
conditions the bx ! ax pathway (Scheme 20) is the main source of
ax (immonium) ions that could dissociate further to form internal
fragments and/or internal immonium ions.
Protonation of the nitrogen of the oxazolone ring leads to
weakening of the –O–CO– bond. Elimination of CO occurs on a
concerted pathway (Paizs et al., 2000) involving rupture of two
covalent 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 b2þ ions was
found to be 29.0, 30.5, 34.6, and 30.3 kcal/mol, respectively. The
bx ! ax TSs are energetically less favored than the separated final
products leading to 3.7, 14.6, 9.6, and 18.4 kcal/mol reverse
activation barriers for the b2 ions listed above (Paizs et al., 2000).
This energetics is in keeping with the experimental fact that
bx ions fragment on the metastable ion time scale by elimination
of CO with substantial KER (Yalcin et al., 1995, 1996),
suggesting that the stable form of bx ions fragment through a
transition structure that is higher in energy than the final products.
It is worth noting here that El Aribi et al. (2003) have recently
shown that the linear a2 ions can be further stabilized by
nucleophilic attack of the N-terminal amino nitrogen on the
carbon of the immonium moiety, forming a five-membered
ring. 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 least
two backbone amide bonds and leads to ions with underivatized
N- and oxazolone or immonium C-terminus. The major
characteristics of amide bond cleavage are described in detail
in the previous sections during the discussion of the bx yz,
a1 yz, etc. PFPs, the same rules apply to the internal fragment
dissociation channels as well. It must be noted that internal
fragments appear in the low-energy collision induced MS/MS
spectra of protonated peptides in mostly those cases when
unusually abundant y ions are present. This situation occurs most
frequently for peptides containing Pro and His (for more details,
see sections on the proline and histidine effects). This finding is in
line with the fragmentation behavior of higher bx ions, which tend
to fragment on the bx ! bx 1 and bx ! ax pathways instead of
cleavage of intact amide bonds.
Ballard & Gaskell (1991) have investigated formation of
internal fragments of protonated H-Tyr–Gly–Gly–Phe–LeuOH and des-Arg1-substance P using sequential product ion
scanning and reaction intermediate scanning experiments. Both
protonated H-Tyr–Gly–Gly–Phe–Leu-OH and des-Arg1-substance P fragment, resulting in abundant internal fragments under
the mass spectrometric conditions applied. For both peptides
certain yz ions show high inherent tendencies to fragment further
to give internal fragments. For example, internal fragments of
des-Arg1-substance P belong to the axy8 and bxy8 classes formed
by cleavage at the Lys–Pro amide bond (proline effect). While
the bx ions are also present in the MS/MS spectrum of protonated
535
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PAIZS AND SUHAI
SCHEME 19.
des-Arg1-substance P, reaction intermediate scans indicate that
the 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 of
protonated peptides can be formed on the a1 yz and b2 ! a1
pathways described above. However, the low-mass region of the
MS/MS spectra often contains immonium ions originating from
amino acid residues located at other positions. Formation of such
internal immonium ions requires cleavage of at least two amide
bonds similarly to the dissociation channels leading to internal
fragments. (Formally, internal immonium ions are (anyN nþ1)1
type internal fragments.)
536
There are two major pathways, which lead to internal
immonium ions (Ix). Ambihapathy et al. (1997) have found that
ax ions—formed on the bx yz and bx ! ax pathways as shown in
Scheme 21—tend to fragment further to ax 1 and Ix ions on the
ax ! ax 1 pathway. In a recent study, El Aribi et al. (2003) have
shown that the ax ! ax 1 pathway can be considered as a suitable
generalization of the a1 yz pathway (Paizs & Suhai, 2001a)
since concerted cleavages of the Ca–CO and OC–N bonds occur
in both cases in the –HCa–CO–NH2þ– and –HCa–CO–NHþ=
moieties, respectively. That is, after repelling out CO a protonbound dimer of ax 1 and the imine of Ix is formed on the
ax ! ax 1 pathway. The dissociation of this proton-bound dimer
is determined by the internal energy distribution of the
fragmenting population and the PAs of the imines formed
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 20.
(Harrison et al., submitted for publication) and the ax 1/Ix ion
abundance ratio can be approximated by using suitable generalization of Equation 2. This mechanism is supported by the
presence of weak ion signals corresponding to loss of CO from
the a2 ions derived from protonated H-Gly–Ala-OH, H-Ala–
Gly-OH, and H-Val–Ala-OH. Also, for many of the investigated
a2 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 immonium
ions are observed with the most abundant being that formed by
proton attachment to the monomer of higher PA.
Another route which leads to internal immonium ions
involves formation of yz ions which fragment further on the
a1 yz pathway as shown in Scheme 21. This mechanism is
supported by metastable ion and low-energy collision induced
dissociation experiments (Ambihapathy et al., 1997) on various
peptides including H-Tyr–Gly–Gly-OH, H-Pro–Gly–Gly-OH,
H-Phe–Leu-OH, etc. which show energetics characteristic of the
a1 yz pathway. It is important to note here that formation of the
Phe immonium ion of protonated H-Tyr–Gly–Gly–Phe–LeuOH can be explained by the [MH]þ ! y2 ! IPhe route (Ambihapathy et al., 1997).
A special case occurs if the y1 ion (protonated amino acid)
dissociates further to the IN immonium ion by eliminating CO
and water. This reaction is of importance for tryptic peptides
since interpretation of the corresponding MS/MS spectra is often
facilitated by assigning the C-terminus to Lys or Arg using the
immonium 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 of
the examples of the amino acid selective fragmentation channels
with 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 the
chemistries behind the proline and other specific effects (like for
example the aspartic acid and histidine effects). The latter are
clearly because of specific chemical activity of the corresponding
side chains, which can be shut down by appropriate chemical
modifications (e.g., esterification of the Asp side chain). On
the other hand, it is the personal view of the present authors that
the proline residues exert their specific activity via affecting the
otherwise non-specific fragmentation pathways in such a way
that leads to dominance of a few ions. These preliminary
considerations are based on investigations currently under way in
our laboratory on a large number of peptides containing proline.
We believe that detailed studies on the bx yz and diketopiperazine pathways of protonated peptides containing proline will
satisfactorily explain the major characteristics of the proline
effect.
Many of the protonated proline containing peptides show
distinct fragmentation behavior producing abundant y ions Nterminal to the proline residues. The proline effect (Schwartz &
Bursey, 1992) was intestigated by CID studies of pentapeptides
containing proline at various positions showing that H-Ala–
Ala–Pro–Ala–Ala-OH and H-Ala–Ala–Ala–Pro–Ala-OH
fragment producing abundant y3 and y2 ions, respectively. The
proline effect was explained by Schwartz & Bursey (1992)
considering the high PA of proline.
Vaisar & Urban (1996) have investigated the CID spectra of
another set of protonated pentapeptides including H-Val–Ala–
Pro–Leu–Gly-OH, H-Val–Ala–NMeAla–Leu–Gly-OH, and
H-Val–Ala–Pip–Leu–Gly-OH. As expected, protonated HVal–Ala–Pro–Leu–Gly-OH produces an abundant y3 ion
whereas both protonated H-Val–Ala–NMeAla–Leu–Gly-OH
and H-Val–Ala–Pip–Leu–Gly-OH fragment mainly to form b3
ions. The behavior of H-Val–Ala–Pip–Leu–Gly-OH is especially striking since the amino acids Pro and Pip differ only by
a –CH2– group. The behavior of H-Val–Ala–NMeAla–Leu–
Gly-OH and H-Val–Ala–Pip–Leu–Gly-OH suggests that
methylation of the amide nitrogen (N-methylation effect)
promotes formation of the b ions of the C-terminal neighbor
amide bond. An explanation of the dramatic difference between
the 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 proposed
that formation of the b ion on the C-terminal side of Pro is not
537
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PAIZS AND SUHAI
SCHEME 21.
favored because of the highly strained [3.3.0] bicyclic moiety
formed. The corresponding H-Val–Ala–Pip–Leu–Gly-OH b3
ion has a less strained [4.3.0] bicyclic moiety and the Nmethylation effect determines the fragmentation. It is worth
noting here that the metastable ion spectrum of protonated HGly–Pro–Gly-OH shows a dominant b2 ion (Ambihapathy et al.,
1997) which contains the [3.3.0] bicyclic moiety. Since other
low-energy fragmentation channels leading to y1 ions are
available for protonated H-Gly–Pro–Gly-OH, statements about
the disfavored energetics of the b ion having the [3.3.0] bicyclic
moiety should be considered cautiously. Computational studies
(Paizs, unpublished data) on the structure and energetics of
538
various fragment ions of H-Gly–Pro–Gly-OH led to similar
conclusions.
The present authors believe that the proline residue has no
specific effect on the cleavage of the N-terminal amide bond of
protonated peptides and the cleavage of such amide bonds can be
described by the rules of the a1 yz pathway. For example, the
results of metastable ion and low-energy CID studies on
protonated H-Val–Pro-OH (Harrison et al., 2000) are in line
with the basic characteristics of the a1 y1 pathway leading to
preference of the y1 ion at low energies (PA of Pro is higher than
that of the imine derived from Val (Paizs et al., 2004)). As
mentioned above, Pro seems to have a rather specific effect when
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
located at the C-terminal of tripeptides. For example, protonated
H-Leu–Gly–Pro-OH (Nold et al., 1997) shows a rather specific
fragmentation pattern in the sense that the majority of the y1 ions
are formed on the ‘diketopiperazine’ pathway whereas other
tripeptides 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 at
Work: Fragmentation of Cyclic Peptides
The fragmentation mechanism of cyclic peptides differs
significantly from that of linear peptides. This is because of the
fact that there is no free terminal amino group in cyclic peptides
and the peptide ring has to be opened prior to fragmentation.
Because of the latter, all sequence ions of protonated cyclic
peptides are originated from secondary cleavages of amide
bonds. Protonated cyclic peptides fragment by loss of CO and
producing sequence ions. Sequence ions of cyclic peptides are
denoted by bxn m and yzn m where the superscript defines the
pair of amino acids between which the backbone opening takes
place. In the following, we briefly summarize a fragmentation
scheme developed recently (Jegorov et al., 2003) for cyclic
peptides based on the bx yz, bx ! bx 1, and bx ! ax pathways.
Fragmentation pathways of a general cyclic pentapeptide
are shown in Schemes 22 and 23. Since cyclic peptides do not
have a free N-terminus, their fragmentation pathways are
dominated by the bx yz channels. The most stable protonation
site (disregarding the basic amino acid side chains) of cyclic
peptides is one of the amide oxygens. Mobilization of the added
proton leads to amide nitrogen protonated species. Ring opening
takes place via oxazolone formation, resulting in a linear peptide
ion having a free N-terminus and an oxazolone ring at the Cterminus (bx yz pathway). The linear ion can fragment by
loosing CO on the bx ! ax pathway, forming a lower b ion
(bx ! bx 1 pathway), or by further mobilization of the extra
proton to an amide nitrogen and subsequent fragmentation on the
bx yz pathways.
The general pentapeptide shown in Schemes 22 and 23
contains a methylated amide nitrogen (aa(3)–aa(4) peptide
bond). As noted in the preceding subsection, N-methylation
induces formation of the b ion of the C-terminal neighbor amide
bond in linear peptides (Vaisar & Urban, 1998). For the cyclic
pentapeptide the corresponding reaction leads to cleavage of the
aa(4)–aa(5) amide bond resulting in a linear peptide as shown in
Scheme 23. The nitrogen of the oxazolone ring of this linear
peptide is methylated, so the ion has a fixed charge. Such ions can
fragment further by reactions specific to the oxazolone ring, for
example, by losing CO on the bx ! ax pathway and by forming a
smaller linear b ion on the bx ! bx 1 pathway (b35–4 in the
present case). It is worth noting here that b35–4 has a mobile
proton since the fixed charge was eliminated in the last step.
These mechanistic considerations can be easily generalized to other cyclic peptides. As a case study, we have
recently explained the fragmentation behavior of protonated
Roseotoxin A (cyclo(-Ile-MeVal-MeAla-beta-Ala-2-hydroxy-4methyl-pentaonyl-3-methyl-Pro-) (Jegorov et al., 2003) based on
these schemes. Some of the mechanistic considerations were
probed by modeling the fragmentation pathways of protonated
Roseotoxin A and by MS/MS experiments.
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III. CHARGE-REMOTE PEPTIDE
FRAGMENTATION PATHWAYS
A. The ‘Aspartic Acid’ Effect
Many of the protonated peptides containing aspartic or glutamic
acid residues show distinct fragmentation behavior producing
abundant b ions C-terminal to these residues. Yu et al. (1993)
have investigated the origin of facile cleavages at Asp–Pro and
Asp-Xxx peptide bonds by MALDI-TOF MS (Xxx denotes other
amino acids). They have found that the Asp-Pro peptide bond is
more labile than the other peptide bonds regardless of the size of
the peptide investigated. The key role of the aspartic acid side
chain in the lability of the Asp–Pro peptide bond has been
demonstrated by esterification of the COOH group of the Asp
side chain which shuts down dominant formation of the b ion Cterminal to Asp. Bakhtiar et al. (1994) have proved that the
activity of the aspartic acid residue depends on the charge state of
the ion under investigation. It was found that cleavage of the
Asp(75)–Met(76) peptide bond in the a-chain of human
apohemoglobin is observed primarily in the 11þ and 12þ
ionization states of the protein. Qin & Chait (1995) have also
observed preferential cleavage of the peptide bonds adjacent to
Asp and Glu residues using a MALDI ion trap mass spectrometer.
Price et al. (1996) have investigated the 11þ ion of ubiquitin
using BIRD. These authors have found that ubiquitin11þ
7þ 4þ
fragments nearly exclusively to produce the b52
/y24 complementary ion pair. This specific fragmentation has been
attributed 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 groups
on a large number of different peptides (Tsaprailis et al., 1999)
under different experimental conditions. The main conclusions
of this work are that nonselective cleavages along the peptide
backbone occur when the number of ionizing protons exceeds the
number of arginine residues (active bx yz pathways), whereas
cleavages adjacent to the acidic residues predominate when the
number of ionizing protons equals the number of arginine
residues (active selective pathways). The latter observation is
explained by the effective sequestration of the added proton(s) by
the side chain of the arginine residue(s) that makes the otherwise
inactive (energetically disfavored if a mobile proton is present)
charge-remote dissociation channels competitive. The chargeremote character of the aspartic acid effect has been proved by
CID studies on peptides containing a fixed charge and Asp
residues (Gu et al., 2000) which behave similarly to peptides
where the number of added protons and the number of arginine
residues are equal.
While our knowledge on the aspartic acid effect was no
doubt enhanced in the last few years, fine energetic and kinetic
details of the underlying processes are still not known. It is now
clear that the COOH group of the Asp side chain takes part in the
enhanced amide bond cleavage but the mechanism is still
uncertain. Also, there are two main possibilities for the structure
of the ions showing pronounced aspartic acid effect. Because the
side chain of Arg contains a functionality of high PA, it is
assumed that these groups are nearly always protonated under the
most common experimental conditions. Therefore, the most
important structure-determining factor for these species is
539
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PAIZS AND SUHAI
SCHEME 22.
‘‘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 terminal
amino group, and the amide oxygens function as electron-donors;
their role is simply solvation of the protonated basic moiety of the
ion by H-bonding. If the acid–base interactions are strong
enough, formation of a SB can take place (Yu et al., 1993; Price
et al., 1996; Tsaprailis et al., 1999) with simultaneous transfer of
the acidic proton of the acidic side chain to other functional
groups of the molecule. The nature of these interactions depends
540
on the amino acids involved, size of the peptide, internal energy
of the ions formed, etc. The mechanisms proposed to account for
the aspartic acid effect involve both CS and SB structures at
various phases of the fragmentation.
The mechanism proposed by Yu et al. (1993) to account for
the aspartic acid effect is shown in Scheme 24a (Path A, compiled
for the case of a general peptide containing both Asp and Arg). It
is assumed that the ‘‘ionizing’’ proton added to the system is
sequestered by the arginine (basic) side chain. In the first step, the
acidic proton of COOH transfers to the nitrogen of the amide
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 23.
bond adjacent to the aspartic acid (Asp) residue forming the
intermediate which contains a SB. Because of the excess energy
deposited on the ion during its formation and excitation, the
Asp-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 bridge
formation between the carboxylate side chain of aspartic acid and
the nitrogen of the adjacent amide bond (i.e., a proton transfer
from the aspartic acid side chain to the neighbor amide nitrogen).
The only difference between the first steps of Paths A and B is that
Price et al. (1996) assume a protonated arginine side chain nearby
stabilizes the SB making it a long-lived intermediate whereas Yu
et al. (1993) contrary to the fact that their peptides also contain
protonated Arg side chains, do not consider such an effect. In the
second step of Path B direct bond cleavage of the amide bond
adjacent to the aspartic acid residue occurs. BIRD experiments
(Price et al., 1996) indicate that the rate limiting step of a possible
complex reaction scheme has a very high frequency factor. This
further suggests an entropically highly favored mechanism such
as a direct bond cleavage of the amide bond adjacent to the
aspartic acid residue. Also, Path B assumes that a relatively long
living intermediate—the SB formed by the side chain of Asp(52)
and the protonated nitrogen of the adjacent amide bond—is
formed in a faster reaction than the dissociation of the SB species.
541
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PAIZS AND SUHAI
SCHEME 24.
542
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
&
SCHEME 24. (Continued )
543
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PAIZS AND SUHAI
SCHEME 25.
A weakness of Path B is that direct bond cleavage of the
nitrogen protonated amide bond leaves behind an extremely
reactive acylium ion. It is clearly not likely that such a reactive
moiety would survive in a large protein. Computational studies
and MS/MS experiments on protonated H-Arg–Asp-NH2 (Paizs
et al., 2002) indicate that another pathway (Path C, Scheme 24c)
operates, where instead of the direct bond cleavage, the Asp-Xxx
peptide bond is cleaved on the corresponding bx yz pathway,
that is by nucleophilic attack of the N-terminal neighbor amide
544
oxygen on the carbon center of the protonated amide bond. This
reaction leads to a b ion with classical oxazolone structure.
A common weakness of mechanisms proposed to account
for the aspartic acid effect based on SB species is that selectivity
of the chemistry is hardly explained. This is because of the fact
that if a SB is formed, the by-product mobile proton (COOH
proton transferred to backbone) could very probably ‘‘visit’’
various backbone protonation sites inducing random fragmentation at the backbone amide bonds. A mechanism that involves
FRAGMENTATION PATHWAYS OF PROTONATED PEPTIDES
only charge solvated species has been recently proposed
(Tsaprailis et al., 1999; Paizs et al., 2002). The low-energy CS
species contain many very flexible torsion angles. For example,
the side chain of aspartic acid can rotate rather freely whenever
the carboxylic group is not involved in solvation of the protonated
guanidine moiety. During its rotation, the hydroxyl group can get
close to the carbon center of the C-terminal neighbor amide bond.
Thus, simultaneous ring formation and transfer of the acidic
proton to the amide nitrogen can occur via a four-center-one-step
mechanism. Such a reaction (Path D, Scheme 24d) results in the
formation of a cyclic anhydride and cleavage of the attacked
amide bond. The attack of the hydroxyl oxygen of the
carboxylate group on the amide carbon is entropically favored
since the reaction results in a five-membered species.
Contrary to recent developments, energetic, kinetic, and
entropy factors determining the activity of the aspartic acid
channels are still not yet fully known. The mechanisms described
above (Path A–D) must be further probed on peptides containing
Asp, varying the length, and the primary sequence of the peptide.
The time-scale of the experiments seems to be also a critical
factor; differences between the activities of Asp and Glu depend
heavily on instrumental setup.
B. Loss of CH3SOH From the Side Chain of Oxidized
Methionine Residues
Methionine oxidation is one of the most frequently occurring
modification to peptides, which is mostly caused by gel electrophoresis sample preparation. The identification of peptides
containing oxidized methionine is facilitated by the characteristic loss of methane sulfenic acid (CH3SOH, 64 Da). Loss of
CH3SOH occurs most dominantly in singly protonated tryptic
peptides indicating that the corresponding elimination reaction is
a charge-remote process. The mechanism of the elimination of
CH3SOH from oxidized methionine has been investigated by
O’Hair & Reid (1999) utilizing MS/MS/MS and deuterium
labeling experiments. Charge directed elimination of CH3SOH
(Scheme 25, route 1) is initiated by mobilization of the extra
proton to form CH3-SOHþ- which is then eliminated by nucleophilic attack of either the Xxx-Met or the Met-Xxx amide
oxygen, leading to formation of a six- or five-membered ring,
respectively. Alternatively, a charge remote mechanism involves
elimination of CH3SOH via 1,2-cis-elimination from the neutral
side chain (Scheme 25, route 2). The labeling and MS/MS/MS
data suggest that the charge remote mechanism is highly competitive with the charge directed PFPs explaining the dominant
loss of CH3SOH from oxidized methionine containing singly
charged trytic peptides.
IV. CONCLUDING REMARKS
This review attempted to summarize the dissociation chemistry
of protonated peptides paying special attention to classification
and characterization of fragmentation pathways leading to
structurally valuable sequence non-sequence ions. It has been
shown that the ‘mobile proton’ model of peptide fragmentation
can be used to understand the MS/MS spectra of protonated
peptides only in a qualitative way rationalizing differences
&
observed for the low-energy fragmentation of peptide ions
having and lacking a mobile proton. To meet all the demands set
forth by the exploding field of proteomics, deeper understanding
of the dissociation chemistry of protonated peptides is needed. To
this end the PIC model that is based on interplay of the major
fragmentation pathways of protonated peptides is proposed.
These fragmentation pathways can be fully characterized to
include all the pre-dissociation, dissociation, and post-dissociation events involved leading to semi-quantative understanding
the MS/MS spectra of protonated peptides. Experimental
and computational data on the fragmentation of protonated
peptides are reevaluated in the light of the PIC model and the
major PFPs.
While some of the PFPs are characterized in quite an
advanced manner there is still a lot to study in the chemistry of
peptide fragmentation. To develop robust IIRs of the MS/MS
spectra of protonated peptides, further investigations are needed.
These studies will involve both the ‘bottom up’ chemical and ‘top
down’ statistical approaches. The present authors’ opinion is that
whereas the ‘bottom up’ approach will provide the necessary
framework (data model) via the PIC model of peptide fragmentation, statistical approaches will be applied to derive the
primary structure related quantitative parameters to be used in the
IIRs.
ACKNOWLEDGMENTS
BP thanks Prof. Alex G. Harrison, Prof. Chrys Wesdemiotis,
Dr. Károly Vékey, Dr. Árpád Somogyi, Dr. Martina Schnölzer,
Dr. George Lendvay, and Dr. István Csonka for many useful
discussions on the chemistry of protonated peptides.
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Béla Paizs received his M.S. and Ph.D. degrees in Chemistry from the Eötvös 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.
Sándor Suhai is head of the Department of Molecular Biophysics at the Deutsches
Krebsforschungszentrum in Heidelberg. He studied physics, mathematics, and chemistry in
Budapest, Göttingen, and Erlangen, respectively, and holds a Ph.D. in theoretical physics
from the Roland Eötvös 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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