RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472

Proton mobility and main fragmentation pathways of

protonated lysylglycine
István Pál Csonka1, Béla Paizs1*, György Lendvay2 and Sándor Suhai1
1
Department of Molecular Biophysics, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
2
Institute of Chemistry, Hungarian Academy of Sciences, P.O. Box 17, H-1525 Budapest, Hungary
Received 27 March 2001; Revised 17 June 2001; Accepted 18 June 2001

Theoretical model calculations were performed to validate the `mobile proton' model for protonated
lysylglycine (KG). Detailed scans carried out at various quantum chemical levels of the potential
energy surface (PES) of protonated KG resulted in a large number of minima belonging to various
protonation sites and conformers. Transition structures corresponding to proton transfer reactions
between different protonation sites were determined, to obtain some energetic and structural insight
into the atomic details of these processes. The rate coefficients of the proton transfer reactions
between the isomers were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) method in
order to obtain a quantitative measure of the time-scale of these processes. Our results clearly
indicate that the added proton is less mobile for protonated KG than for peptides lacking a basic
amino acid residue. However, the energy needed to reach the energetically less favorable but±from
the point of view of backbone fragmentation±critical amide nitrogen protonation sites is available in
tandem mass spectrometers operated under low-energy collision conditions. Using the results of our
scan of the PES of protonated KG, the dissociation pathways corresponding to the main
fragmentation channels for protonated KG were also determined. Such pathways include loss of
ammonia and formation of a protonated a-amino-e-caprolactam. The results of our theoretical
modeling, which revealed all the atomic details of these processes, are in agreement with the
available experimental results. Copyright # 2001 John Wiley & Sons, Ltd.

Protonated peptides activated under low-energy conditions
dissociate mainly by charge-directed fragmentation along
their backbone.1±5 In many cases these reactions result in
structurally informative bn and Yn@ ion series.6±10 (bn and Yn@
ions are formed by charge retention on the N- or C-termini,
respectively.) It is well known from the results of molecular
orbital calculations3±5 that protonation on the amide nitrogen
leads to considerable weakening of the amide bond. On the
other hand, protonation on the amide oxygen makes the
amide bonds even stronger than those in the neutral
counterparts. It is also well known that protonation on the
amide nitrogen is thermodynamically unfavored compared
to other protonation sites like the amide oxygens, the N-
terminal amino group, or basic amino acid (AA) side chains
such as arginine and lysine. In short, from the point of view
of decomposition, protonation on the amide nitrogen is
favorable, while from the thermodynamic point of view this
site is not the most favored one. To explain why decom-
position still occurs, the `mobile proton' model1,3,4,11±20 was
introduced, which proposes that the proton(s) added to a
peptide migrate(s) amongst protonation sites prior to
fragmentation provided that they are not sequestered by a
*Correspondence to: B. Paizs, Department of Molecular Biophy-
sics, German Cancer Research Center. Im Neuenheimer Feld
280, D-69120 Heidelberg, Germany.
E-mail: B.Paizs@DKFZ-Heidelberg.de
Contract/grant sponsor: Hungarian Scienti®c Research Fund;
Contract/grant number: OKTA T22824.
basic AA side chain. The mobile proton model has been
verified experimentally by using deuterium-labelling tech-
niques13,15,17 which indicated strong H/D mixing prior to
collisionally induced dissociation (CID) of MD‡ ions of a
number of small peptides. In a very early study, Tsang and
Harrison11 showed that facile H/D mixing occurs in the D2
and CD4 chemical ionization of AAs. In a recent study
Harrison and Yalcin17 suggested that the proton added to
non-basic AAs, and to peptides containing no basic AAs,
samples all positions bearing labile hydrogens prior to
fragmentation of the protonated species. Also, Wysocki and
co-workers4,14,16 have demonstrated that the relative posi-
tions of fragmentation efficiency curves obtained by electro-
spray ionization in combination with surface induced
dissociation (ESI-SID) depends on the amino acid composi-
tion (absence or presence and type of a basic residue) and on
the sequence and the size of the peptide investigated. This
dependence could be firmly rationalized based on the
`mobile proton' model, providing further support for the
underlying mechanistic considerations.
In our previous ab initio and RRKM studies19,20 we
investigated the rate of proton transfer and isomerisation
processes (transitions determining the `proton traffic' of a
protonated species) in protonated N-formylglycineamide,
glycylglycine, and N-formylglycyl glycineamide, using pure
theoretical methods. These calculations started with a careful
investigation of the potential energy surfaces (PESs) of the
above species to determine minima corresponding to various

DOI:10.1002/rcm.388 Copyright # 2001 John Wiley & Sons, Ltd.

1458 I. P. Csonka et al.
Scheme 1. Various protonated forms of KG. Since the positive charge is delocalized
and the bond orders are not integer numbers, the valence bond limiting structures as
drawn are only approximate representations.
protonation sites and conformers, and transition structures
which describe possible transitions between these minima.
Because all the processes involved in the `proton migration'
are unimolecular in nature, one can estimate the correspond-
ing rate constants by using the RRKM formalism. In the
RRKM calculations we applied the results of quantum
chemical calculations such as energetics, vibrational fre-
quencies, etc.
The most important conclusions of these studies are as
follows. The global minima on the PESs of protonated N-
formylglycineamide and glycylglycine are the isomers
protonated at the amide oxygen and terminal amino
nitrogen, respectively. The relative energies of the minima
corresponding to the other isomers are higher, but the
differences are not so large that they could prevent formation
of isomers protonated at the amide oxygens (protonated GG)
and amide nitrogens, in mass spectrometers operated under
the most common conditions. Detailed analysis of the saddle
points separating various minima on the PES indicate that
low-energy pathways exist which connect the global mini-
mum with potential wells corresponding to isomers proto-
nated at the amide nitrogen. RRKM calculations suggested
that transitions occurring on these pathways are fast and
take place easily on the time-scale of commonly used
spectrometers. These proton transfer (PT) reactions are
usually very fast as soon as the internal energy of the ion
exceeds the corresponding threshold energies. Furthermore,
in most cases, the rates of the PT reactions and of the
conformational transitions around flexible bonds are com-
parable. In the case of a longer peptide chain lacking strongly
basic amino acid residues, the most possible route for proton
migration along the backbone involves proton movement
between adjacent C=O-H¼O=C bridges. Our studies in-
dicated that the rate of proton transfer along the backbone of
the peptide chain by proton hops between the oxygens of
adjacent amide bonds is also fast. Overall, our theoretical
Copyright # 2001 John Wiley & Sons, Ltd.
modeling of proton migration in protonated peptides lends
strong support to the mechanistic considerations involved in
the `mobile proton' model.
As a side project we examined the role of various five-
membered ring structures in the fragmentation of proto-
nated N-formylglycineamide (loss of ammonia) and glycyl-
glycine (loss of water). In these studies we found that the
relative energies of the species containing five-membered
rings are close to those of the amide-nitrogen-protonated
species. Furthermore, the barriers to dissociation initiated
from the five-membered ring structures are high (the
corresponding unimolecular rate constants are small),
indicating that these species are not involved in backbone
fragmentation of the protonated peptides investigated. On
the other hand, we have presented strong theoretical
evidence5,21 which suggests that backbone fragmentation
indeed involves amide-nitrogen-protonated isomers.
The available experimental information on proton migra-
tion in lysine-containing peptides is quite limited. Wysocki
and co-workers investigated4,16 fragmentation efficiency
curves determined by ESI-SID for various peptides contain-
ing lysine, such as leucine-enkephalin analogs, Ax oligopep-
tides substituted by K, etc. These authors found that the
peptides lacking any basic AAs fragment more easily than
those containing either K or R. Also, the lysine-containing
peptides fragment more easily than those containing R. In
the language of the `mobile proton' model this means that
the energy required to produce the fragmenting (reactive)
amide-N-protonated species increases for the series of
peptides containing no basic AA, a lysine, and an arginine
side chain, respectively.
Fragmentation of protonated lysine and small peptides
containing lysine was studied by Harrison and co-work-
ers.22,23 Protonated lysine fragments almost exclusively by
loss of ammonia under both metastable and low-energy CID
conditions. Studies of protonated [a-15N]lysine showed that
Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly 1459

Table 1. Calculated total and relative energies (with ZPE correction) of the investigated conformers of protonated KG at the B3LYP/6-
31G(d) level of theory and the main structure determining interactions. The relative energies are measured from the global minimum of
protonated KG (E01)

Species Energy (a. u.) Rel. Ezpe (kcal/mol) Interactions

Conformers protonated on the e-amino group

E01 705.446754 0.0 Ne-H¼Oamide, Ne-H'¼Na, Namide-H¼Ocarboxyl; delocalization
E02 705.447191 0.1 Ne-H¼Oamide, Ne-H'¼Ocarboxyl=O, Namide-H¼Na
E03 705.446779 0.2 Ne-H¼Oamide, Ne-H'¼Ocarboxyl=O, Namide-H¼Na
E10 705.440298 3.3 Ne-H¼Oamide, Namide-H¼Na, Namide-H¼Ocarboxyl=O; delocalization
E11 705.441123 3.5 Ne-H¼Oamide, Namide-H¼Ocarboxyl=O; delocalization
E12 705.440202 3.5 Ne-H¼Oamide, Namide-H¼Na, Namide-H'¼Ocarboxyl=O; delocalization
E16 705.437334 4.9 Ne-H¼Na, Na-H¼Oamide, Namide-H¼Ocarboxyl; delocalization
E17 705.436847 5.3 Ne-H¼Na, Na-H¼Oamide, Namide-H¼Ocarboxyl=O; delocalization
E18 705.434768 7.2 Ne-H¼Oamid, Namid-H¼Ocarboxyl=O; delocalization
E19 705.429751 8.7 Ne-H¼Na, Na-H¼Oamid, Namid-H¼Ocarboxyl OH; delocalization
E20 705.426675 11.2 Ne-H¼Na, Na-H¼Oamid, Namid-H¼Ocarboxyl OH; delocalization
Conformers protonated on the a-amino group
A01 705.440327 3.5 Na-H¼Ne, Na-H'¼Oamid, Namid-H¼Ocarboxyl=O; delocalization
A02 705.439854 3.5 Na-H¼Ne, Na-H'¼Oamid, Namid-H¼Ocarboxyl=O; delocalization
A03 705.439418 3.7 Na-H¼Ne, Na-H'¼Oamid, Namid-H¼Ocarboxyl=O; delocalization
A10 705.432689 7.7 Na-H¼Ne, Na-H'¼Oamid, Namid-H¼Ocarboxyl OH; delocalization
A13 705.430559 9.4 Na-H¼Ne, Na-H'¼Oamid, Namid-H¼Ocarboxyl OH; delocalization
A15 705.427518 11.4 Na-H¼Oamid, Namid-H¼Ne, Namid-H¼Ocarboxyl=O; delocalization
A20 705.423985 14.6 Namid-H¼Ne, Na-H¼ Ocarboxyl=O, Na-H'¼Oamid
A21 705.411258 21.8 Namid-H¼Ne, Na-H¼ Ocarboxyl=O
A22 705.402464 27.3 Ocarboxyl OH-H¼Ne, Na-H¼ Ocarboxyl=O
Conformers protonated on the amide oxygen
O01 705.426279 11.3 Namid-H¼Ne, Oamid-H¼Na, Ne-H¼Ocarboxyl=O,Namid-H¼Ocarboxyl=O
O02 705.416834 17.5 Namid-H¼Ne, Oamid-H¼Na, Ne-H¼Ocarboxyl OH,Namid-H¼Ocarboxyl OH
O03 705.414935 18.8 Namid-H¼Na, Oamid-H¼Ocarboxyl=O, Na-H¼Ne
O08 705.410184 21.2 Oamid-H¼Na, Na-H¼Ne, Namid-H¼Ocarboxyl=O
O14 705.401433 26.6 Namid-H¼Na, Oamid-H¼Ocarboxyl=O
O15 705.401361 27.0 Oamid-H¼Ocarboxyl OH, Namid-H¼Ne, Na-H¼ Oamid
O16 705.400817 27.3 Namid-H¼Ne, Oamid-H¼Ocarboxyl OH
Imine-type conformers, protonated on the e-amino group and on the amide oxygen
I01 705.426910 11.5 Ne-H¼Nimine, Ne-H'¼ Ocarboxyl=O, O(amid)-H¼Na, delocalization
I02 705.413858 19.9 Ne-H¼Na, Ne-H'¼Ocarboxyl=O
I03 705.408663 23.1 Ne-H¼Nimine, Ne-H'¼ Ocarboxyl=O, Na-H¼O(amid), delocalization
Conformers protonated on the amide nitrogen
D01 705.408436 22.5 Ê
Namid-H¼Ne, Namid-H'¼Na, Na-H¼Ocarboxyl=O;Camid-Ocarboxyl=O 2.812 A
D02 705.406218 24.1 Ê
Namid-H¼Ne, Namid-H'¼Na, Na-H¼Ocarboxyl=O;Camid-Ocarboxyl=O 2.839 A
D03 705.395373 30.8 Ocarboxyl OH-H¼Ne, Namid-H¼Na, Namid-H'¼Ocarboxyl=O
D04 705.394236 31.4 Namid-H¼Na, Na-H¼Ne; Camid-Ocarboxyl=O 2.747 A Ê
D06 705.393793 31.8 Namid-H¼Na, Namid-H'¼Ocarboxyl=O, Na-H¼Ne,
D10 705.388727 34.9 Namid-H¼Na, Namid-H'¼Ocarboxyl=O, (Camid-Ne, 3.713 A Ê)
D15 705.388367 35.7 Namid-H¼Na, Namid-H'¼Ocarboxyl=O, Camid-Ne, 2.665 AÊ
D16 705.385700 36.6 Namid-H¼Na, Namid-H'¼Ocarboxyl=O, (Camid-Ne, 3.827 A Ê)
D17 705.386395 36.7 Namid-H¼Na, Namid-H'¼Ocarboxyl=O, Camid-Ne, 1.999 AÊ
D18 705.385779 36.7 Namid-H¼Na, Namid-H'¼Ocarboxyl-OH, Na-H¼Ne,
D19 705.386396 36.7 Namid-H¼Na, Namid-H'¼Ocarboxyl=O, Camid-Ne, 2.704 AÊ

the expelled ammonia specifically carried away the nitrogen
of the side chain in both unimolecular (i.e., collison-free) and
collision-induced fragmentation. Harrison and co-workers
suggested that the ion formed by ammonia loss from
protonated K is protonated pipecolic acid. Amongst the
investigated peptide derivatives, protonated KG was found
to have main dissociation pathways leading to loss of
ammonia and formation of an ion with m/z 129. Although
the m/z 129 ion is nominally an acylium ion, its metastable
ion characteristics and CID spectrum suggest that this ion is
indeed a protonated a-amino-e-caprolactam. The formation
of the m/z 129 ion was found to be characteristic for other
lysine-containing peptides as well.
Copyright # 2001 John Wiley & Sons, Ltd.
In the present work we extend our theoretical modeling of
proton migration in peptides to include lysylglycine, which
is one of the simplest peptides containing a basic amino acid.
Our goal here is to validate the mobile proton model using a
combined ab initio/RRKM methodology for peptides con-
taining a basic amino acid. Furthermore, a scan of the PES of
protonated KG enables us to calculate the energetics and
kinetics of the main fragmentation channels of this ion. In the
remainder of this paper, after summarizing the methods
used, we present the results pertaining to the proton transfer
in protonated lysylglycine (KG), followed by the results
related to the mechanism of decomposition via ammonia
loss and a-amino-e-caprolactam formation.
Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
1460 I. P. Csonka et al.

COMPUTATIONAL DETAILS
In the case of molecules which contain many flexible
torsional degrees of internal freedom, generating the proper
input geometries is at least as important as the choice of the
employed theoretical method. In the last few years we
worked out an efficient search engine20,21,24 which can
explore the low-energy structures of protonated peptides
in a rather efficient way. Our strategy involves molecular
dynamics simulations and subsequent geometry optimiza-
tions at both low (initial ab initio scan) and higher levels (final
ab initio scan) of theory. The whole system is highly
Figure 1. Equilibrium structure of E01, the global minimum of
automated using scripts and small programs. The search
protonated lysylglycine.
applied for protonated KG resulted in species protonated at
the side-chain e-amine group (E, Scheme 1), species proto-
nated at the a-amine group (A, Scheme 1), species
naming scheme as follows. The first character in the acronym
protonated at the carbonyl oxygen of the amide bond (O,
refers to the protonation site (Scheme 1). In the names, the
Scheme 1), species protonated at the nitrogen atom of the
letters E, A, O, and D denote isomers protonated on the e-
amide group (D, Scheme 1), and species which contain an
amine N atom, on the a-amine N, on the amide oxygen and
imine bond instead of the K-G amide bond (I, Scheme 1). The
on the N atom involved in the amide bond, respectively. The
ab inito calculations were carried out at the HF/3-21G level of
I conformers contain imine bonds instead of the amide bond
theory for the initial scan of the PES of protonated KG.
connecting the K and G units. The second and third
In our previous articles19,20 we showed that the results of
characters, 01, 02, 03, etc., denote respectively the most
RRKM calculations depend much less on the level of theory
stable, second most stable, etc., investigated minimum for a
employed for the quantum chemical calculations than the
given structural isomer. For example, E01 represents the
calculated relative energies of the conformers do. This
most stable species among the conformers in the series
observation allows us to use a relatively inexpensive
protonated on the e-amine group of the lysine residue. The
quantum chemical level, so that the final ab initio scan of
notations of the proton transfer TSs are constructed from
the PES of protonated KG was carried out at the B3LYP/6-
those of the corresponding minima, with the lower mini-
31G(d) level of theory in the present work. The ab initio
mum occupying the first place. For example, E10_D01
calculations were carried out using the Gaussian 98 program
represents a TS connecting E10 and D01, where E10 has
suite.25
lower energy than D01.
After checking the structures obtained at the B3LYP/6-
The calculated total and relative energies of selected
31G(d) level to be true minima by calculating harmonic
conformers of protonated KG are collected in Table 1. The
frequencies, we located transition states (TSs) which connect
relative energies are measured from the global minimum of
different protonation sites or correspond to the main
the PES of protonated KG, isomer E01 in our nomenclature,
fragmentation pathways of protonated KG. The TSs found
and are corrected for ZPE. The main stabilizing interactions
were checked to be first-order saddle points by calculating
of the conformers are also given in Table 1. Our search for the
harmonic frequencies. Furthermore, in some cases, we
minima on the PES of protonated KG resulted in a very large
applied IRC (intrinsic reaction coordinate) calculations to
number (approximately 200) of various species. This is due
check which minima are connected by a given TS. The zero
to the numerous possible protonation sites of the neutral
point energy (ZPE) corrections were calculated from the
molecule and also to the many flexible rotational degrees of
harmonic frequencies with no scaling factor.
freedom such as those of the lysine side chain. It is evident
Using the results of the aforementioned ab initio calcula-
that a detailed description and visualization of such a
tions, the rate coefficients for the transitions between the
tremendous amount of information is not possible. There-
minima on the PES were calculated using the RRKM
fore, we describe only those structures in detail which are
method,26,27 over a grid of energies up to 120 kcal/mol of
internal energy. We used the same method for each type of
transition (proton transfer or fragmentation transition). No
tunneling correction was included for two reasons: (1) in
most cases the rates are very high as soon as the energy is
above the reaction threshold; and (2) the accuracy of a
few kcal mol 1 for the barrier height does not warrant the
attempt to achieve the accuracy addressed by a tunneling
correction.

RESULTS AND DISCUSSION

Conformers of protonated KG
The conformers of protonated KG are identified using the Figure 2. Equilibrium structure of A01.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly
Figure 3. Equilibrium structure of O01.
energetically favored and, accordingly, the data presented in
tables are restricted to those isomers which are important
from the point of view of any (PT or fragmentation) reaction.
The global minimum on the PES of protonated KG is one
of the conformers of the e-amine protonated isomer, namely,
E01 (Fig. 1). In this conformer the positively charged e-amine
group is stabilized by H-bonds, Ne-H¼Oamide and Ne-
H'¼Na, and the hydrogen of the amide bond is involved in a
Namide-H¼Ocarbonyl interaction. Furthermore, in E01, the
glycine residue is planar, indicating a possible conjugation
over this part of the molecule. The calculated energies of the
next two conformers in the E-series are almost the same as
that of E01 (Table 1). In E02 the positively charged e-amine
group is stabilized by two strong H-bonds, one of them
involves the oxygen of the amide-bond (Oamide), while the
other involves the carbonyl oxygen of the C-terminal
carboxyl group (Ocarbonyl). A third H-bond is present
between the hydrogen of the amide bond and the a-amine
group (Na). The structure of E03 is very similar to that of E02.
The most stable conformer amongst the Ax structures, A01
(Fig. 2), is 3.5 kcal/mol less stable than E01. In this species
the positively charged a-amine group is stabilized by H-
bonds Na-H¼Ne and Na-H'¼Oamide. Furthermore, the
hydrogen of the amide bond is involved in the Namide-
H¼Ocarbonyl H-bond, and there is a possible conjugation
over the glycine residue due to the planarity of this part of
Figure 4. Equilibrium structure of I01.
Copyright # 2001 John Wiley & Sons, Ltd.
1461
Figure 5. Equilibrium structure of D01, the starting point of the
fragmentation reaction over TS_B1_B.
the molecule. In the next two conformers of the Ax series,
A02 and A03, the same interactions are present as in A01,
and, consequently, they have only a slightly higher relative
energies (3.5 and 3.7 kcal/mol, respectively). In our opinion
the higher energies of the Ax structures relative to those of Ex
can mainly be attributed to the larger proton affinity of the e-
amino group, but the larger ring strain of the rings created by
H-bonds in the Ax-structures also plays some role in the
relative stability of the Ex and Ax species. Although both
protonated amine groups can be stabilized by two strong H-
bonds, the e-amine group is positioned at the end of a long
and flexible alkyl chain while the a-amine group is
connected to the a-carbon of the relatively rigid amide bond.
This causes a larger ring strain for the Ax species especially
in the case of the H-bonds involving Oamide. For example, in
the Ex-structures, one of the H-bonds is involved in a nine-
membered floppy ring while a similar interaction for the Ax
structures induces formation of a five-membered ring. The
different bonding pattern has a significant impact on the
geometric parameters of the respective Na-H¼Oamide H-
bonds. For example, while the H¼Oamide distance is 1.701 A Ê
and the Ne-H¼Oamide angle is 156.11 ° in E01, at the same
time H¼Oamide is 1.862 A Ê and Na-H¼Oamide is 121.33 ° in
A01. These features clearly indicate that the Na-H¼Oamide
interaction is weaker in A01 than in E01. In general, the
energy ranges of the ten most stable conformers of Ax and of
Ex overlap.
The equilibrium energy of the most stable conformer of
KG protonated at the carbonyl oxygen, O01 (Fig. 3), is
11.3 kcal/mol above E01. Its structure is very similar to that
of O1 of protonated GG.20 In both cases the protonated
Oamide is stabilized by a H-bond involving Na and there is
also a possibility for a conjugation over the backbone of the
molecules due to their planarity. For O01 (protonated KG)
Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
1462 I. P. Csonka et al.

Table 2. Calculated absolute and relative energies (with ZPE proton donor. In conclusion, it is not the case that Ox
correction) of the investigated PT TSs of protonated KG structures are destabilized, but that the Ax/Ex structures are
calculated at the B3LYP/6-31G(d) level of theory. The relative overstabilized in KG compared to the case of GG, due to the
energies are with respect to the global minimum of protonated larger extent of intramolecular solvation of the positively
KG (E01) charged center. The O02 conformer possesses the same main
features as O01, although its glycine moiety is not planar. In
TS Energy (a. u.) Rel. Ezpe (kcal/mol)
contrast, the structure of O03 is quite different from the
A01_E16 705.436901 3.4 former Ox structures. This species is stabilized by Oamide-
A04_E17 705.436382 3.8 H¼OcarboxylO, Namide-H¼Na and Na-H¼Ne H-bonds.
A10_E18 705.429682 7.5
When scanning the conformational space of protonated
A13_E20 705.426546 9.8
E02_O14 705.401422 26.5 KG we found some minima (Ix, Scheme 1) which contain
A15_O01 705.422469 11.8 imine bonds instead of the K-G amide one. The Ix structures
A13_O08 705.410100 20.1 can be considered as tautomers of the Ex structures (Scheme
I01_O01 705.423424 11.1 1). All the Ix species were obtained from geometry
I03_O16 705.397879 26.9
optimizations started from structures protonated on the
E10_D01 705.408079 21.1
E12_D02 705.405789 22.7 amide oxygen. In some cases when the e-amino group was
A21_D01 705.396606 27.9 close to the hydrogen of the amide nitrogen, spontaneous
A20_D02 705.396137 28.2 transfer of this proton to the basic e-amino group occurred.
A22_D03 705.387146 34.0 In this way the former protonated amide oxygen became a
hydroxyl group, and these species are protonated at the e-
amine group. The relative energies of structures of this type
the hydrogen of the amide group is involved in a H-bond are comparable to those of the Ox structures. In fact, the
with the e-amine group. This means that the O01 conformer relative energy of I01 (Fig. 4) is only 11.5 kcal/mol, which is
of KG is stabilized by one more intramolecular interaction almost as low as that of O01. In I01 the positively charged e-
than the O1 of GG. In spite of this extra stabilizing effect, the amine group is stabilized by H-bonds, Ne-H¼Nimine and Ne-
relative energy of O1 of protonated GG with respect to the H'¼Ocarbonyl, while the H-bond O(amide)-H¼Na and the
most stable a-amine-protonated isomer is 1.2 kcal/mol,20 possible conjugation over the planar backbone of the
while that of O01 is 7.8 kcal/mol. An investigation of the molecule provide further stabilization for the molecule. In
structures of the A isomers of protonated KG and GG shows conformer I02 the H-bonds Ne-H¼Na and Ne-H'¼OcarboxylO
the reason why O01 seems to be less stable: there is efficient imply stabilization for the positively charged e-amine group,
extra stabilization of the A isomers of protonated KG with while there is no H-bond involving the hydroxyl group
respect to those of GG. While in GG the Ax structures formed from the amide oxygen and also there is no
(including A1, the global minimum) contain only one strong possibility for the conjugation since the backbone of the
H-bond involving the positively charged amine group, the molecule is not planar. This could be the reason for its
lowest energy Ex and Ax structures of KG are stabilized by 19.9 kcal/mol relative energy which is much higher than that
two strong H-bonds. In contrast, the Ox structures cannot of I01. Conformer I03 has 23.1 kcal/mol relative energy, and
take advantage of the larger number of H-bond acceptor the main interactions are Ne-H¼Nimine, Ne-H¼Ocarboxyl=O,
groups in KG compared to the case of GG, because the Na-H¼O(amide) and a possible conjugation over the planar
protonated Oamide can participate only in one H-bond as a backbone of the molecule.

Table 3. ZPE-corrected barrier heights in kcal/mol of the PT reactions relative to the connected lower (left columns) and higher (right
columns) energy minimum and unimolecular rate constants for reaction starting from the respective minima calculated at selected internal
energies by the RRKM method. (Note that lower estimates for the rate are given for processes with a ‘negative’ barrier, see text for
details.)

Lower minimum Barrier log(k1ev) log(k32) Log(k2ev) TS Barrier log(k1ev) log(k32) log(k2ev) Higher minimum

A01 0.1 v.f.a v.f.a v.f.a A01_E16 1.5 v.f.a v.f.a v.f.a E16
A04 0.1 v.f.a v.f.a v.f.a A04_E17 1.6 v.f.a v.f.a v.f.a E17
A10 0.2 v.f.a v.f.a v.f.a A10_E18 1.2 v.f.a v.f.a v.f.a E18
A13 0.4 v.f.a v.f.a v.f.a A13_E20 1.3 v.f.a v.f.a v.f.a E20
E02 26.4 Ð Ð 3.5 E02_O14 0.1 Ð v.f.a v.f.a O14
O01 0.5 12.2 12.4 12.5 O01_A15 0.4 12.2 12.3 12.4 A15
A13 10.7 3.7 7.6 9.4 A13_O08 1.1 v.f.a v.f.a v.f.a O08
O01 0.2 v.f.a v.f.a v.f.a O01_I01 0.4 v.f.a v.f.a v.f.a I01
I03 3.8 Ð 9.7 11.3 I03_O16 0.4 Ð v.f.a v.f.a O16
E10 17.9 Ð 3.1 5.6 E10_D01 1.3 v.f.a v.f.a v.f.a D01
E12 19.2 Ð 1.7 4.5 E12_D02 1.4 Ð v.f.a v.f.a D02
A21 6.1 Ð 7.3 9.5 A21_D01 5.4 Ð 8.2 10.3 D01
A20 13.6 Ð 3.8 7.3 A20_D02 4.1 Ð 9.1 10.9 D02
A22 6.6 Ð Ð 9.3 A22_D03 3.2 Ð Ð 10.7 D03
a
Very fast.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly
The lowest energy species protonated on the amide N,
D01 (Fig. 5), has 22.5 kcal/mol energy relative to E01. This
species is stabilized by the Namide-H¼Na and Namide-H¼Ne
H-bonds and charge-transfer interaction between Ocarbonyl
and the protonated amide bond. The main difference
between the most stable amide-nitrogen-protonated isomers
of GG and KG is that the latter is stabilized by an extra H-
bond between the protonated amide nitrogen and the e-
amine group. Otherwise, the Dx structures of these two
species are very similar to each other. The additional
stabilizing H-bond is probably the reason why, compared
to the Ox structures, D01 of KG has lower energy than the
corresponding D1 species of GG (11.2 kcal/mol vs.
15.7 kcal/mol). However, as compared to the global mini-
mum, the relative energy of D01 is larger than that of the
similar D1 species of GG at 16.9 kcal/mol.20 The D02
conformer has a similar structure to D01, and accordingly
its relative energy is just a little higher at 24.0 kcal/mol. The
other Dx structures have much higher relative energies. This
finding can be explained by the fact that in those structures a
positively charged amide nitrogen is stabilized by weaker H-
bonds than those of D01 and D02. For example, conformer
D03 has 30.8 kcal/mol relative energy, and in this structure
the amide nitrogen is involved in the H-bonds Namide-H¼Na
and Namide-H¼Ocarbonyl (an Ocarboxyl OH-H¼Ne H-bond is
also present).
Proton mobility in protonated lysylglycine
In our previous studies19,20 on protonated N-formylglycina-
mide, N-formylglycyl glycinamide and diglycine, we found
that most of the internal rotations connecting different
conformers of a given protonated structure are very fast.
They occur on a much shorter time-scale than the MS
experiments even at relatively low internal energy values.
Therefore, during the evaluation of proton mobility for
protonated KG, we focused our attention on the PT reactions
and did not deal in detail with the very large number of
internal rotations connecting the different conformers of a
given protonated structure. The calculated total and relative
energies of the investigated PT transition states (TS) of
protonated KG are collected in Table 2. The barrier heights
are measured from the global minimum of the PES of
protonated KG, isomer E01 in our nomenclature. The ZPE-
corrected barrier heights relative to the respective minima
are collected in Table 3, together with the rate constants
calculated using the RRKM method at internal energies of
1eV (23.06 kcal/mol), 2eV (46.12 kcal/mol), as well as at
32.1 kcal/mol, which is the threshold energy of the lowest-
energy fragmentation pathway of protonated KG (see
below).
The data presented in Table 3, however, require a caveat.
Our combined DFT/RRKM model of proton transfer
reactions assumes that the investigated protonation sites
are separated by substantial barriers, i.e. the relative energy
of any TS is higher than those of either of the minima
connected by that TS. In such a case one could apply ab initio
data to obtain kinetic information on the given process by
using the RRKM formalism which provides internal-energy-
dependent unimolecular rate constants. The potential sur-
face of protonated KG involves a number of potential
Copyright # 2001 John Wiley & Sons, Ltd.
1463
minima which, formally, prove not to be minima when the
ZPE of the vibrational modes is also taken into account. This
means that the region of the configuration space correspond-
ing to such isomers is not a basin in which the system can be
trapped. Instead, formally it can be considered as a shoulder
or small plateau on the wall of a deeper well. We think,
however, that as the minima were unequivocally detected by
the minimum search, it is better to consider them as distinct
minima when their energy differs significantly because it
seems to be reasonable that such structures exist, and in
addition the vibrational ZPE, which is conventionally
calculated from separable harmonic modes, is not accurate
enough to guarantee full accuracy. (However, more accurate
calculation of ZPE by including factors like anharmonicity,
mode-mode coupling etc., is far beyond the reach of any
theoretical method for a molecule of this size). It is a
question, then, of how one can calculate the rate of a reaction
to and from such a poorly defined isomer: the quantum
chemical calculation yields a `negative barrier' for such
reactions, which is clearly nonsense. We distinguish two
types of such cases:
1 The two minima on the PES have similar relative energies
and the relative energy of the TS connecting them becomes
smaller when ZPE correction is applied than those of the
minima themselves. In this case the proton is shared by the
two protonation sites and essentially oscillates between the
bridgehead atoms in a shallow potential energy valley
instead of a double well potential corresponding to two
clearly separated isomers. This means that the concept of
reaction rate does not apply to the virtual reactions. When
the PES of a protonated peptide is represented by many
such valleys, the most important transitions are internal
rotations connecting these shallow valleys along perpen-
dicular directions. Our previous studies19,20 showed that
these internal rotations around flexible bonds are fast.
2 One of the corresponding minima is at a much lower
energy level than the other, and the relative energy of the
TS connecting them becomes smaller than that of the
higher minimum after the ZPE correction is applied. This
situation corresponds to the physical picture in which the
proton is shared by the two protonation sites, but it is more
localized on the lower energy site and can be found only
with a small probability in the proximity of the higher
energy site. Being a fast process, an internal rotation can
occur while the proton is close to the higher energy
protonation site and this process can result in a structure in
which the proton is really localized on that site. It is also
possible that a real chemical reaction can occur while the
shared proton stays close to the higher energy protonation
site, which is usually more reactive than the low-energy
species. As we assume here that the higher energy
minimum exists, but we obtain from the quantum
chemical calculation a `negative barrier height', we can
only estimate the rate. We assume that, although the TS
after ZPE correction does not correspond to an energetic
barrier, it still can present a `bottleneck' in phase-space on
the way from one protonation site to another. As the ZPE-
corrected barrier is always just a little below the energy of
the higher minimum (and the difference is actually in the
Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
1464 I. P. Csonka et al.

order of the accuracy of the calculation itself), in the RRKM respective Ax structures. The Ax → Dx reactions became
calculations we consider the energy of the TS to be equal to relatively fast at less than 1.5 eV. For example, the relative
that of the higher minimum. This means that reaction from energy of the A21_D01 TS is 27.9 kcal/mol above E01 but
the higher energy minimum is extremely fast while a only 6.1 kcal/mol above A21, so that the rate constant is
reasonable estimate for the rate of the reverse process can 2  107 s 1 already at 32.1 kcal/mol internal energy. Com-
be obtained. paring the accessibility of the amide nitrogen protonation
site in protonated GG and KG, one notes that transfer of the
After thus describing the underlying theoretical background, added proton to this site needs significantly larger energies,
we turn to the detailed discussion of proton traffic in and is correspondingly much slower for the case of KG. This
protonated KG. The most favored protonation sites of is clearly due to the presence of a basic side-chain group
protonated KG are the e- and the a-amino groups, as capable of forming low-energy amino-protonated isomers in
discussed in the previous section. Although the relative KG, which makes the relative energy of the amide-nitrogen-
energies of the Ax structures tend to be slightly higher then protonated species rather high compared to the analogous
those of the Ex structures, the differences are not very large. protonated GG. This has a significant effect on the
We found some low barriers corresponding to TSs A01_E16, fragmentation of lysine-containing peptides, making these
A04_E17, A10_E18, A13_E20 which, in principle, connect the molecules more resistant to backbone fragmentation com-
Ax and Ex basins, but each proves to disappear after the ZPE pared to other non-basic peptides.
correction is applied. This means that the proton motion in
the molecule is so fast that there seems to be no prolonged
Main fragmentation pathways of protonated KG
existence of either of these isomers. In other words, the Ax to
Protonated KG fragments mainly on two dissociation path-
Ex and the reverse reactions are very fast even at low internal
ways on the metastable ion time scale.23 These two pathways
energies.
are also important for other lysine derivatives and lysine-
As we showed earlier,20 the Ox structures play an
containing peptides. The first reaction is loss of ammonia
important role in the mobility of the added proton, since
(57% of the total ion signal in the metastable ion fragmenta-
most probably the amide oxygens are heavily involved in the
tion spectrum), while the second one is formation of an ion
transfer of the proton along the peptide chain. There is a TS
with m/z 129 (40% of the total ion signal). Interestingly,
connecting the Ex/Ax manifold of minima with the O-
neither the a1 ion (NH2‡=CH-(CH2)4-NH2 immonium ion)
protonated isomers: passage through TS A15_O01 is
nor the y1 ion (protonated glycine) are formed during low-
extremely fast in both directions even at low internal
energy dissociation of protonated KG, although these types
energies. We located another barrier (E02_O14) which
of fragment ions are prominent products in the mass spectra
connects E02 with a high-lying Ox conformer (a shoulder
of other dipeptides.28±30
in terms of the previous discussion), but the corresponding 15
N-Labeling experiments22 on the fragmentation of
transition is quite slow according to the results of the RRKM
protonated lysine have shown that the ammonia lost
calculations and cannot be considered to be an efficient
specifically involves the nitrogen of the side chain. This
pathway. It is possible that there are further fast channels
reaction produces an ion with m/z 130, and this ion
connecting the Ex/Ax and the Ox conformers.
fragments further resulting in an ion with m/z 84. Based on
The lowest-lying conformers of the Ix and Ox isomers
the stoichiometry and the kinetic energy release (KER) of this
(both at around 11.5 kcal/mol above E01) are connected by
second fragmentation (m/z 130 → 84), Yalcin and Harrison
TS I01_O01. The barrier to this reaction disappears after
suggested that the ion with m/z 130 in the mass spectrum of
correcting for ZPE indicating a very fast transition for which
protonated lysine and other lysine derivatives may be a
we did not carry out RRKM calculations. Other Ix_Ox
protonated pipecolic acid23 (Scheme 2).
transitions start at significantly larger energies (for example,
The calculated absolute and relative energies of the
TS I03_O16 is at 26.9 kcal/mol relative energy), and become
structures occurring on the fragmentation paths leading to
fast only at higher internal energies. Considering both the
loss of ammonia and formation of an ion at m/z 129 from
kinetic and energetic information available for the Ix species,
protonated KG are collected in Tables 4 and 5, and the results
one can conclude that formation of these species is very
are visualized in Fig. 6. The calculated rate constants obtained
likely in mass spectrometers operated under the most
by RRKM theory are shown in Table 6 and in Fig. 7.
common conditions.
Based on the experimental observation of Dookeran et al.22
As mentioned above, fragmentation of backbone amide
on protonated lysine, we assumed that ammonia loss from
bonds requires protonation of the corresponding amide
nitrogen. Therefore, PT reactions connecting the Dx valleys
with others play an important role in formation of the
reactive configuration for these decompositions. Our theo-
retical modeling suggests that direct Ex $ Dx reactions have
a large activation energy and are quite slow even at large
internal energies. For example, E10_D01 connects the Ex and
Dx wells at 24.5 kcal/mol relative energy, and the rate
constant is as small as 4  105 s 1 even at 2 eV internal
energy. The transition between the Ax and Dx structures is Scheme 2. Protonated pipecolic acid, the supposed product ion
more facile, mainly due to the higher internal energy of the of the ammonia loss from the side chain of protonated lysine.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly 1465

Table 4. Calculated total and relative energies (including ZPE correction) of the TSs and products (molecular complexes) of the
investigated fragmentation reactions of protonated KG calculated at the B3LYP/6-31G(d) level of theory. The relative energies are
measured to the global minimum of protonated KG (E01)

TS Energy (a. u.) Rel. Ezpe (kcal/mol) Complex Energy (a. u.) Rel. Ezpe (kcal/mol)

Ammonia loss
TS_NH3_Na 705.391923 32.1 CO_NH3_Na 705.434911 5.9
TS_NH3_O 705.38536 35.9 CO_NH3_O 705.414198 17.6
b1 ion formation (caprolactam formations)
TS_B1_A 705.382850 39.3 CO_B1_A 705.420364 15.8
TS_B1_B 705.382319 39.4 CO_B1_B 705.420103 15.9
`a1-y1' pathway
TS_A1Y1 705.358446 51.0 CO_A1Y1 705.391440 29.0

Table 5. Calculated absolute and relative (kcal/mol, including ZPE correction) energies of the neutral and ionic fragmentation products of
protonated KG

resp. complex a.u a.u Rel. to E01 Rel. to the complex Rel. to the resp. TS

Ammonia loss
Prot. pipecolic acid der. NH3
CO_NH3_Na 648.872782 56.547948 13.3 7.4 18.8
Prot. 7-membered ring NH3
CO_NH3_O 648.852710 56.547948 25.0 7.4 11.0
b1-ion formation
a-amino-e-caprolactam Glycine
CO_B1_A 420.976601a 284.420042 29.6 13.8 9.8
CO_B1_B 420.976601a 284.420042 29.6 13.7 9.8
a
Protonated at the amine group.

Table 6. ZPE-corrected barriers of the TSs of the investigated fragmentation reactions relative to the reactant minimum and unimolecular
rate constants at selected internal energies calculated by the RRKM method

Minimum TS Barrier Log(k2eV) Log(k3eV) Log(k4eV)

Ammonia loss
E11 TS_NH3_Na 28.6 2.7 6.3 8.0
E03 TS_NH3_O 38.5 Ð 4.0 6.1
b1-ion formation (caprolactam formations)
D17 TS_B1_A 2.6 10.1 11.1 11.3
D01 TS_B1_B 16.9 3.6 7.5 8.8
`a1-y1' pathway
D01 TS_A1Y1 28.5 Ð 4.5 7.7

protonated KG involves the e-amino group. That is, our involved in such a reaction: (a) the a-amino group, (b) the
search for various minima and transition structures occur- amide O, (c) the carbonyl oxygen of the carboxyl group, and
ring during loss of ammonia from protonated KG was (d) the hydroxyl oxygen of the carboxyl group. According to
initiated from species protonated at the side chain of the our calculations, the last two cases can be ruled out as
lysine residue (Ex structures). The most straightforward important processes, because the TSs and the products of
possibility would be a direct cleavage of the Ce-Ne bond, these reactions have very high relative energies (>55 kcal/
which results in a carbocation. The calculations showed, mol). Hence we will consider only those cases where the a-
however, that this process requires a substantial amount amino group or the amide oxygen substitutes Ne, resulting in
of energy (more than 60 kcal/mol relative to the global
minimum E01). In addition, the carbocation structure is not
stable: it is transformed to isomers with a cyclic structure,
without the need to pass a barrier, in intramolecular
reactions in which one of the nucleophilic groups of the
molecule (for example a-amine or amide O) attacks the
carbocation center and forms the ring. This led us to assume
that bond rupture between Ce and Ne atoms and attack of a
suitable nucleophile on the positive center occur in a
concerted manner (SN2-type reaction, Scheme 3). There are Scheme 3. Ammonia loss from the protonated lysine side chain
four nucleophilic groups in protonated KG, which can be through an SN2-type reaction.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
1466 I. P. Csonka et al.

Figure 6. Calculated energetics of the investigated fragmentation reactions of protonated KG.

Figure 7. RRKM rate constants of the investigated fragmentation reactions of protonated KG.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly 1467

Scheme 4. SN2-type substitution reaction on Ce involving the a-amino group resulting in a

protonated pipecolic acid derivative and ammonia.

The transition structures obtained for these pathways,

denoted as TS_NH3_Na for path a and TS_NH3_O for path
b, are shown in Figs 8 and 9, respectively. In the case of
TS_NH3_Na the nitrogen of the a-amino group attacks the
Ce atom. As seen in Fig. 8, the Ne±Ce distance is lengthened
Ê ) while N1 gets close to Ce (2.140 A
(2.067 A Ê ) and the CeH2

Scheme 5. SN2-type substitution reaction on Ce involving the group becomes planar. In the case of TS_NH3_O the amide
amide O resulting in a protonated seven-membered ring oxygen attacks the Ce center (Fig. 9). Similarly to the TS on
path a, cleavage of the Ce-N bond (2.133 AÊ ) and planarity of
(imidocaprolactone) derivative and ammonia.

a molecular complex formed by the leaving ammonia and a

pipecolic acid derivative (path a, Scheme 4) or a seven-
membered ring derivative (path b, Scheme 5), respectively.

Figure 10. Equilibrium structure of CO_NH3_Na.

Figure 8. Transition structure TS_NH3_Na.

Figure 9. Transition structure TS_NH3_O. Figure 11. Equilibrium structure CO_NH3_O.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
1468 I. P. Csonka et al.

the CeH2 group show direct evidence for an intramolecular

SN2-type reaction. Also, the short distance between atoms
Oamide and Ce (1.993 AÊ ) indicates that formation of the seven-
membered ring is nearly completed. According to an
investigation of the geometries on the product wing of the
IRC, the products of these reactions are molecular complexes Scheme 6. b1-ion formation resulting in a molecular complex of
formed by ammonia and a pipecolic acid derivative neutral glycine and protonated a-amino-e-caprolactam.
(CO_NH3_Na, Fig. 10) along path a, and an aminocapro-
lactone imide (CO_NH3_O, Fig. 11) along path b. The
relative energy (5.9 kcal/mol) of CO_NH3_Na is lower by fore, these authors concluded that the ion with m/z 129 is an
approximately 12 kcal/mol than that of CO_NH3_O a-amino-e-caprolactam formed by a ring-closure coincident
(17.6 kcal/mol). This difference is due to the fact that with the expulsion of a neutral glycine23 (Scheme 6).
CO_NH3_Na is a pipecolic acid derivative protonated at a Since substantial evidence exists5,29,30 that cleavage of
secondary amine. On the other hand, in CO_NH3_O, amide bonds requires protonation on the amide nitrogen, we
protonation occurs on an imide nitrogen. initiated our search for the pathway leading to formation of
The relative energies of TS_NH3_Na and TS_NH3_O are the protonated a-amino-e-caprolactam from the Dx species.
very close to each other at 32.1 and 35.9 kcal/mol, These calculations resulted in two TSs, namely TS_B1_A
respectively. However, the unimolecular rate constants (initiated from D01) and TS_B1_B (initiated from D17)
calculated by RRKM theory show that reaction via path a shown in Figs 12 and 13, respectively. Protonation on the
is faster, indicating that formation of a pipecolic acid amide nitrogen makes the carbon of the amide bond even
derivative is more probable in the mass spectrometer. Both more positive than the neutral counterpart. In this way, this
reactions are relatively slow and reach the ms characteristic
time at approximately 3 and 4 eV internal energy, respec-
tively. An experimental confirmation of the theoretical
prediction can be based on the difference of the products
of paths a and b: further fragmentation of the pipecolic acid
derivative will be different from that of the caprolactone
derivative. This means that an MS/MS experiment on the
fragment ion of the ammonia loss can decide which path
dominates the ammonia loss of lysine derivatives, or both
can play a role in this process. Path a corresponds to the
fragmentation pathway suggested by Yalcin et al., namely,
that the ion at m/z 130 is a pipecolic acid derivative. On the
other hand, reactions similar to that including TS
TS_NH3_O can also play a major role in the substantial
ammonia loss observed for lysine derivatives lacking the a-
amino group, such as Ac-Lys-OH (11% in metastable
fragmentation23) or Ac-Lys-OMe (32% in metastable frag-
Figure 12. Transition structure TS_B1_A.
mentation23). This latter type of reaction can also be involved
in the ammonia loss of such peptides, where the lysine
residue is not positioned at the N-terminus but somewhere
in the chain or at the C-terminus (for example a peptide
resulting from a tryptic digest).
The second main fragmentation pathway of protonated
KG results in a b1 ion (m/z 129) (40% relative intensity in the
metastable ion spectrum23). It is well known that a-
aminoacylium ions are unstable and exothermically elim-
inate CO.31 In fact, there is only one known example for an
detectable b1 ion, namely, that formed from an N-terminal
methionine residue of peptides, which is supposed to have a
cyclic structure involving the S atom of the methionine side
chain.32 On the other hand, bn (n 2) ions of larger peptides
are known to contain a protonated oxazolone-type ring.5,31
Taking into account this information, and the fact that the
m/z 129 ion derived from protonated KG is stable, one can
expect that it has a structure other than an acylium ion.
Yalcin and Harrison23 showed that the metastable fragmen-
tation of the ion with m/z 129, and also the kinetic energy
release values measured from the metastable peaks, are very
similar to those of protonated a-amino-e-caprolactam. There- Figure 13. Transition structure TS_B1_B.

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly 1469

Figure 16. Equilibrium structure of CO_B1_B.

Figure 14. Equilibrium structure of D17, the reactant of the
fragmentation reaction over TS_B1_A.
described above, a substantial difference is observed
between the unimolecular rate constants calculated for the
carbon becomes rather electrophilic and can be attacked by
two pathways. For the entire energy interval investigated the
nearby nucleophilic groups. In a similar situation most of the
pathway including TS_B1_B is much faster than that
protonated dipeptides29,30 fragment via concerted cleavages
including TS_B1_A. This difference can be explained by
of the amide and the Ca-Camide bonds, since there is no
the fact that, while the e-amino group is heavily involved in
nucleophilic center in the proximity of the protonated amide
the stabilization of the protonated amide bond for D01 (Fig.
bond which could stabilize the large partial positive charge
5), this is not the case for D17 (Fig. 14), where the e-amino
by ring formation. However, in the case of protonated KG,
group is located above the plane of the amide bond taking
the e-amino group is close enough to the protonated amide
part in stabilization by a weaker charge-transfer interaction.
bond, and can be involved in such a stabilization (for
One has to note, however, that the relative energy of D17 is
example see Figs 5 and 14). The analysis of the IRCs shows
definitely higher than that of D01. Therefore, it is very likely
that reactions via TS_B1_A and TS_B1_B start from two
that the population of the D17 isomer is much lower than
different isomers, D01 and D17, respectively, and result in
that of D01 in mass spectrometers operated under usual
two ion-molecule complexes of a caprolactam derivative and
conditions. On the other hand, if the fast fragmentation
glycine, CO_B1_A and CO_B1_B, respectively (Figs 15 and
reaction empties this state but the PT reactions can feed it
16, respectively). It is very interesting that, although the
from other isomers, i.e. if the fragmentation is rate
structures of CO_B1_A and CO_B1_B are quite different,
determining, then this pathway may be an efficient channel
their relative energies are very similar at 15.8 and 15.9 kcal/
for the formation of a caprolactam derivative. This may
mol, respectively. On the other hand, the energies of the
happen at high excitation energies. If, however, re-popula-
initial conformers are significantly different, i.e. D17 is
tion of D17 is relatively slow, as is indicated in the
14.2 kcal/mol less stable than D01.
calculations presented in the previous section, the resulting
The relative energies of TSs TS_B1_A and TS_B1_B are
rate of reaction is slower so that the ammonia-loss reaction
essentially the same at 39.3 and 39.4 kcal/mol, respectively.
discussed above can successfully compete with caprolactam
This, however, does not mean equal reaction rates. If one
formation and result in comparable abundance ratios.
assumes that the reaction starts from the initial isomers
Interestingly, the y1 ion (protonated glycine) is not present
in the metastable ion spectrum of protonated KG.23 In recent
papers we investigated the mechanism of y1-ion formation
from protonated GG29 and other dipeptides.30 A new
pathway (`a1-y1') was proposed which leads to integrated
formation of a1 and y1 ions, the ratio of which depends on the
composition and the energy distribution of the fragmenting
species for a particular dipeptide. The first TS in this

Scheme 7. First step of the ‘a1-y1’ fragmentation pathway of

Figure 15. Equilibrium structure of CO_B1_A. dipeptides.29,30

Copyright # 2001 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
1470 I. P. Csonka et al.
Scheme 8. Proton transfers in molecular complexes CO_B1_A.
mechanism corresponds to concerted cleavage of the amide
and the Ca-Camide bonds resulting in a trimer of a protonated
imine, the C-terminal amino acid and CO (Scheme 7). After
loss of CO, a proton-bound dimer of the a1 ion and the C-
terminal amino acid is formed. Under low-energy conditions
the lifetime of this dimer is long enough that numerous
proton transfers can take place between the imine and amino
acid in the dimer. There is a pathway through which the
dimer can dissociate without passing a barrier in the next
step (depending on the energy of the ion, the PA of its
monomers, etc.) to form either a1 or y1 ions.
We explored the initial step of this `a1-y1' pathway in the
case of protonated KG as well. The calculation revealed that,
on the reaction path starting from the D01 structure, the
corresponding saddle point, TS_A1Y1, with a relative
energy of 51.0 kcal/mol, is the highest barrier among all
the TSs we found to lead to fragmentation. The product of
this reaction also has a high relative energy of 29.0 kcal/mol
(Table 4, Fig. 6). Furthermore, according to the RRKM
calculations, this is the slowest fragmentation reaction at
low internal energies, and it approaches the rate of the
concurrent reactions only at very high internal energies
(Table 6, Fig. 7). We did not explore further this complicated
reaction pathway, as the key steps are presumably similar to
those found in the case of protonated GG.29
It is clear that the formation of the b1 ion in the case of
protonated KG is favored compared to the `a1-y1' pathway at
low internal energies, totally depleting the a1 and y1
channels. However, a generalization of the `a1-y1' mechan-
ism to the case of protonated KG, where the complex formed
in the fragmentation reaction containing a protonated a-
amino-e-caprolactam instead of the a1 ion of other peptides,
leads one to expect that y1-ion formation could also take
place via proton transfer from the amide-nitrogen-proto-
nated a-amino-e-caprolactam to the neutral glycine in
CO_B1_A and CO_B1_B (Figs 15 and 16, respectively). At
first sight this process seems to be very favorable because an
amide N protonation site, based on experience on the
relative stability of isomers of dipeptides, is expected to be
less favorable than the amino group of G. However, a closer
Copyright # 2001 John Wiley & Sons, Ltd.
look at the structures CO_B1_A and CO_B1_B (Figs 15 and
16, respectively) reveals that there are serious reasons for the
a-amino-e-caprolactam to remain protonated. First, the
proton transfer to glycine in CO_B1_A and CO_B1_B would
result in a trans-amide bond in the a-amino-e-caprolactam,
which causes a high ring strain in the seven-membered ring.
Second, the other proton of the protonated amide nitrogen
can transfer to the a-amino group of the a-amino-e-
caprolactam itself, resulting in a cis-amide bond causing
much less ring strain (Scheme 8). Our calculations indicate
that decomposition of CO_B1_A to form the amide-nitro-
gen-protonated a-amino-e-caprolactam plus G requires
28.0 kcal/mol energy, while decomposition to trans-a-ami-
no-e-caprolactam plus protonated G requires 34.6 kcal/mol.
It is striking that the third pathway, i.e. formation of the
amino-protonated a-amino-e-caprolactam plus G, requires
only 13.9 kcal/mol energy. This energetic argument explains
why y1-ion formation does not take place for protonated KG
under metastable conditions. However, it is possible that a
protonated dipeptide KX, having a C-terminus amino-acid
residue with higher proton affinity than that of glycine,
could fragment to produce y1 ions. For example, the proton
affinity of valine can be large enough to compete with the a-
amino site of the a-amino-e-caprolactam. An MS experiment
on protonated lysylvaline (KV) would be decisive on this
question.
Finally, we compare the energetics (Fig. 6) and kinetics
(Fig. 7) of the main fragmentation pathways of protonated
KG leading to ammonia loss and formation of the protonated
a-amino-e-caprolactam (b1 ion). As can be seen in Fig. 6, the
pathways leading to loss of ammonia are energetically
favored compared to those resulting in formation of proto-
nated a-amino-e-caprolactam (b1 ion). On the other hand,
unimolecular rate constants calculated using RRKM theory
formally prefer formation of the a-amino-e-caprolactam (b1
ion) (Fig. 7). However, pathways leading to the formation of
the b1 ion are initiated from Dx conformers, while ammonia
loss occurs from the Ex isomers. In protonated KG the PT
processes re-populating the Dx isomers are relatively slow so
that, at low excitation, this may be the rate-determining step.
Rapid Commun. Mass Spectrom. 2001; 15: 1457±1472
 Proton mobility and fragmentation pathways of protonated Lys-Gly
Scheme 9. Proton traffic in protonated lysylglycine involving the
most important protonation sites (E, A, O and D, see text).
Numbers set in roman denote the energies of the minima, while
numbers in italic denote the height of the barriers between them
(all values calculated at the B3LYP/6-31G(d) level of theory and
are ZPE-corrected). Bold letters are the acronyms for the given
protonation sites in our nomenclature.
In the present case we do not have all the barriers on all
possible paths connecting all isomers that lead from the
Ex/Ax basin to the Dx basin, so that we cannot provide a
numerical estimate of the relative importance of ammonia
loss and caprolactam derivative (b1 ion) formation. However,
the energetic and kinetic data obtained in our calculations do
provide a qualitative understanding of the main fragmenta-
tion pathways for protonated KG.
CONCLUSIONS
The theoretical model of the dynamical processes in
protonated KG (Scheme 9) presented in this work involves
a detailed understanding of the potential surface of the ion
based on density functional theory (B3LYP/6-31G(d)) and
estimation of the time-scale and relative rates of the
isomerization and fragmentation processes using RRKM
calculations. We found five structural isomers in which the
proton is located on the amino nitrogen of the e-position (E),
the a-position (A), on the carbonyl oxygen of the carboxyl
group (O), on the N atom participating in the amide bond
(D), as well as a tautomer of E (I). The structures and
energies of numerous conformers of these were determined.
The E and A isomers were found to be the most stable, with
relatively small potential barriers separating the potential
wells of their conformers as well as connecting the E and A
basins. The O, D, and I isomers are in higher regions of the
potential surface, and in many cases are dynamically not
well defined because the potential barriers separating them
from the deep A and E minima are very low. Our DFT/
RRKM modeling showed that the accessibility of the reactive
amide-nitrogen-protonated species is less pronounced for
protonated KG than for other peptides lacking basic
residues. This observation is in line with the experimental
results showing that lysine-containing peptides fragment
less easily than protonated peptides lacking any basic AAs.
Copyright # 2001 John Wiley & Sons, Ltd.
1471
The fragmentation reactions of protonated KG take place
in very interesting complex, concerted processes. Direct
chain breakage was found to be much less favored than the
concerted processes resulting in cyclic products. One group
of pathways leads to the loss of ammonia from the e-amino
group and formation of either a protonated pipecolic acid
derivative or a protonated imido derivative of an a-amino-e-
lactone. The other group of decomposition pathways starts
from the amide-nitrogen-protonated isomer, and results in
the formation of a glycine molecule and a protonated
aminocaprolactam (b1-ion). Interestingly, the `a1-y1' frag-
mentation pathway, which is important in protonated GG,
does not play a role in the fragmentation of protonated KG.
The theoretical calculations firmly identify the structures of
the possible fragmentation products, and make possible the
qualitative understanding of the metastable ion spectrum of
protonated KG.
Acknowledgements
Financial support of this work by the Hungarian Scientific
Research Fund (G.L, OTKA T 22824) is gratefully acknowl-
edged.
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