Selective deletion of the internal lysine residue from the peptide sequence by collisional activation

Article abstract of DOI:10.1007/s13361-012-0456-1

The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue. American Society for Mass Spectrometry, 2012.

Full text of DOI:10.1007/s13361-012-0456-1

B American Society for Mass Spectrometry, 2012
J. Am. Soc. Mass Spectrom. (2012) 23:1967Y1980
DOI: 10.1007/s13361-012-0456-1
RESEARCH ARTICLE
Selective Deletion of the Internal Lysine Residue
from the Peptide Sequence by Collisional
Activation
Shibdas Banerjee, Shyamalava Mazumdar
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India
Abstract
The gas-phase peptide ion fragmentation chemistry is always the center of attraction in
proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we
describe the formation of an anomalous fragment ion, which corresponds to the selective
deletion of the internal lysine residue from a series of lysine containing peptides upon collisional
activation in the ion trap. We detected several water-loss fragment ions and the maximum
number of water molecules lost from a particular fragment ion was equal to the number of lysine
residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine
residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine
functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-
terminal α-amino acetylation had no effect on this process indicating involvement of the side
chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by
multistage CID of the modified and unmodified peptides, by isotope labeling and by energy
resolved CID studies. The results suggest that the lysine deletion might occur through a
unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate
formed by the loss of water from a lysine residue in the protonated peptide. This intermediate
subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide
backbone leading to the deletion of an internal lysine residue.
Key words: Synthetic peptide, Electrospray ionization, Collisional activation, Water-loss, Internal
lysine deletion, Cyclic imine intermediate
proteins in biofluids, especially in a small quantity of
complex mixture [4]. The sequences of individual peptides
Introduction
recise determination of the sequence of amino acids in a
protein is important for protein profiling such as in the
study of the roles of specific proteins in diseases and for the
discovery of potential biomarkers as well as of targets for
therapy of diseases [1–4]. Mass spectrometry-based methods
have become extremely popular for high throughput assay of
produced by enzymatic digestion of a protein are derived
from the identification of the gas-phase peptide ion frag-
ments by collision-induced dissociation (CID) in tandem
mass spectrometry (MS/MS) [5–9].
The mass selected protonated peptide undergoes frag-
mentation on collisional activation and the success of the
tandem mass spectrometric identification of the peptides
depends on the understanding of the mechanism of frag-
mentation of the peptide ions. The gaseous peptide frag-
mentation model based on cleavage of the peptide backbone,
is now implemented in various sequencing algorithms to
analyze the MS/MS spectra of peptides [10]. However, only
P
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-012-0456-1) contains supplementary material, which
is available to authorized users.
Correspondence to: Shyamalava Mazumdar; e-mail: shyamal@tifr.res.in
Received: 1 May 2012
Revised: 15 July 2012
Accepted: 20 July 2012
Published online: 25 August 2012

1968 S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides
a small fraction of the acquired ion signals in the MS/MS
spectrum of a peptide can be assigned unambiguously by the
existing sequencing programs. The majority of the signals in
the tandem mass spectrum of a peptide ion still remain
unassigned. When a gaseous protonated peptide is collision-
ally excited, it follows several complicated dissociation
pathways and only a few of them are well studied. So the
understanding of the mechanism of the formation of any
anomalous fragment ion that cannot be assigned by the
conventional model of peptide dissociation would no doubt
help to improve and modify the sequencing algorithms used
in proteomics.
The collisional excitation of the protonated peptide leads
to transfer the ionizing proton from an unreactive site of
higher gas-phase basicity (e.g., Arg, Lys, and His side chain
or N-terminal α-NH2 group) to energetically less favored and
reactive backbone-amide(s) and carbonyl(s) in the mobile
proton model [11, 12]. Fast proton transfer to various
backbone amide bonds results in the formation of the
primary fragment ions e.g. N-terminal a-, b-, c-ions and C-
terminal x-, y-, and z-ions [9, 13] that are used for sequence
identification of the peptide. Under low energy collision
condition, a, b and y-fragments are abundant. The b-ion was
proposed to exist as cyclic oxazolone structure and/or head-
to-tail macrocyclic structure in the gas phase [14–16].
Increase in the activation time (i.e., in multiple collision
condition), the primary fragment ions can further dissociate
to secondary ions (satellites) such as internal fragments [17],
amino acid specific immonium ions [18], neutral loss a/b/y-
ions (e.g., bn – NH3 or bn – H2O etc.) [19, 20] and smaller
member of a-, b-, and y-ion series. All these ions are directly
related to the sequence of the peptide and hence they have
been referred to as direct sequence ions [21].
However, the majority of the fragment ions detected in
the MS/MS spectrum of a peptide often cannot be classified
as direct sequence ions [22–25]. These fragments are formed
by complex rearrangements of the ions and termed as
nondirect sequence ions [21]. There are a few such nondirect
sequence ions have been identified recently, such as
scrambled fragments of the b/a type ions resulting in the
loss of an amino acid residue from the interior of the peptide
chain [21, 26]. The first step of such loss of an internal
amino acid by sequence scrambling fragmentation pathway
was proposed [21, 27–32] to involve a nucleophilic attack
by N-terminal α-NH2 group on the acylium carbocation of
the b-ion (a primary fragment) forming a protonated cyclic-
peptide intermediate (CPI). This CPI subsequently under-
goes ring opening at different amide bonds producing
sequence scrambled linear b-ions. These scrambled linear
b-ions undergo further dissociation by conventional frag-
mentation pathway to produce corresponding daughter ions
that are not related to the original sequence of the peptide
[21, 27–32]. Thus such ions, called the nondirect sequence
ions, would be related to all the possible scrambled
sequences of the peptide, and therefore would loss the
memory of the original sequence. At the first stage of CID,
this unique fragmentation of the cyclic b-ion is difficult to be
observed.[21, 33, 34] However under multi stage CID
(MSn), these b-ions (containing 3–9 amino acid residues)
were shown to follow the above scrambled fragmentation
pathways [21, 27–32, 35, 36]. The propensity of the above
sequence scrambling increases with the increase in the chain
length and charge states of the peptides [37].
In this report, we present a detailed study of the gas-phase
fragmentation of a series of synthetic octapeptides of
different sequences containing six alanines and two lysines.
The results show that the internal lysine residues of these
peptides are selectively lost upon collisional activation. The
mechanism of deletion of the internal lysine residue has been
investigated by multi stage CID, chemical, and isotopic
modification of the peptides and energy resolved CID
techniques. Our results suggest that this selective deletion
of the lysine residue from the interior of the peptide may not
follow the sequence scrambling fragmentation pathway as
reported earlier [21, 27–32]. A novel gas-phase peptide ion
degradation pathway has been shown to be responsible for
the specific loss of the internal lysine residues in the peptide.
Experimental
Chemicals
Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, and 2-(1-H-
benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluoro-
phosphate (HBTU) were obtained from Novabiochem (Merck,
Hohenbrunn, Germany). Fmoc-Rink amide MBHA resin LL
(100–200 mesh), triisopropylsilane (TIS), methyl tert-butyl
ether (MTBE), and GC grade methanol were purchased from
Merck. DMF, piperidine, N-methylmorpholine (NMM), trietha-
nolamine, acetic anhydride, and formaldehyde were provided by
S.D. Fine-Chem Ltd. Mumbai, India. Borane pyridine complex
(~8 M BH3) and Fmoc-Lys(Boc)-OH-13C6, 15N2 (98 atom %)
were obtained from Sigma-Aldrich, St. Louis, MO, USA. HPLC
grade acetic acid and trifluoroacetic acid (TFA) were purchased
from Spectrochem Pvt. Ltd. Mumbai, India.
Synthesis of C-Terminal Amidated Peptides [38]
Standard Fmoc solid-phase peptide synthesis technique was
employed to prepare resin-bound linear peptides AKAAA
KAA, AAAKKAAA, KAAAKAAA, KAAAAAAK,
AAAKAAA, AKAAAK*AA (the sixth lysine residue is
isotopically labeled) and YAKGFKL, either manually or in
batch mode on an automated peptide synthesizer (PS3 by
Protein Technologies, Inc. Arizona, USA) at 0.1 mmol scale
using a fourfold excess of Fmoc-amino acids relative to
Fmoc-Rink amide MBHA resin (0.39 mmol/g). Deprotection
was performed using 20 % piperidine/DMF. Coupling was
performed using 1:1:2 amino acid/HBTU/NMM in DMF.
DMF top washes (1 min) were performed between depro-
tection and coupling steps. The peptides were cleaved from

S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides 1969
resin using 2.5 % triisopropylsilane (TIS), 2.5 % water,
95 % TFA solution at room temperature for 3 h. The
volatiles were removed under a stream of nitrogen gas, and
peptides precipitated with methyl tert-butyl ether. Then it
was centrifuged and peptides were collected as trifluoroace-
tate salt. The peptides were purified by reversed-phase high-
performance liquid chromatography (RP-HPLC).
Low-energy CID spectra (MS/MS and MSn) were
acquired using an isolation width of 2m/z unit. The
activation Q (as labeled by Thermo Finnigan, used to adjust
the qz value [10] for the precursor ion) was set at 0.250, and
the activation time employed was 30 ms at each CID stage.
The normalized collision energy (which defines the
amplitude of the RF energy applied to the end-cap electro-
des in the CID experiments) was set between 25 % and
30 % for multi stage CID experiments (MSn) and it was
varied from 10 % to 55 % for the dissociation profile
(breakdown) study of the mass selected ions. Data acquisition
was performed for 1 min using XCalibur software (Thermo
Fisher Scientific).
Selective N-Terminal α-Aminoacetylation
of the Peptides [38]
The resin bound peptides (where the N-terminus α-NH2 is
free but ε-NH2 of lysines are BOC protected) obtained from
the above procedure was allowed to react with 100-fold
excess acetic anhydride in the same fashion as above amide
bond formation. This led to the selective acylation of the N-
terminus α-NH2 of the peptides. The acetylated peptides
were then cleaved from the resin and ε-NH2 of lysines were
deprotected following the same process as above to get
target N-terminal α-aminoacetylpeptides. The product was
purified by RP-HPLC.
Results and Discussion
The ESI-MS spectra of all the isomeric octapeptides
(AKAAAKAA-NH2, AAAKKAAA-NH2, KAAAKAAA-
NH2, KAAAAAAK-NH2, AAAKAAAK-NH2) produced
singly protonated species [M+H]+ at m/z 700.5 and doubly
protonated species [M+2 H]+ at m/z 350.8 in the gas phase
(see Figure S1 in the Supporting Information). Collisional
activation (MS/MS) of the singly protonated peptide [M+H]+
(m/z 700.5) produced the characteristic bn and yn ions (see
Figure 1). Figure 1 shows that the major fragment ion (base
peak) is the b8-ion (at m/z 683.5), which is produced by the loss
of NH3 from the C-terminal protonated carboxamide functional
group of the peptides.
Reductive Methylation of the Amine Functional
Groups in the Peptides [39]
Peptides (1.25 mg) were dissolved in 1 mL 300 mM
triethanolamine buffers (pH 7.5). The reactions were diluted
to 1 mg/mL with methanol. Pyridine-BH3 (8 M in excess
pyridine) was added to 30 mM. Last, formaldehyde was
added to 20 mM. Reactions were then sonicated briefly in a
water bath (1 min) to suspend pyridine-BH3 and allowed to
react at room temperature for 2 h. Then it was lyophilized
and the products were taken for mass spectrometric experi-
ments without further purification.
Neutral Loss of Water
(a) Correlation with the Number of Lysine Residues.
A close inspection of all the MS2 spectra (Figure 1)
shows that the sequence informative ions (bn and yn)
undergo neutral loss of one or two water molecules.
The maximum number of water-loss from a bn-ion is
found to be equal to the number of lysine residues
present in that ion. For example, the maximum
number of water loss observed from b8-ion of
AKAAAKAA-NH2 is two, while only one water-loss
is observed from b5, b4, and b3-ion of this peptide.
This observation clearly correlates the number of
water loss with the number of lysine residues in the bn-ion,
suggesting that the lysine residues are involved in the
water-loss mechanism.
ESI-MS Experimental Condition
Electrospray ionization mass spectrometry (ESI-MS) studies
[10, 40] were carried out using a Thermo Finnigan LCQ
Deca (San Jose, CA, USA) Electrospray quadrupole ion trap
mass spectrometer [38, 41–43]. All the experiments were
carried out under the identical conditions. The flow rate of
the analyte solution was maintained at 5 μL/min. Nitrogen
was used as the sheath gas. The ion source conditions were:
sheath gas flow rate 20 units (arbitrary for the Finnigan
systems, corresponding to ~0.375 L/min). Capillary (des-
olvation) temperature was maintained at approximately
200 °C and capillary voltage was kept at 15 V. The ion-
spray voltage and tube lens offset were maintained at 4.5 kV
and –7 V respectively. The ion optics were tuned by using
autotune routine within the LCQ Tune program to get
maximum ion count. The sample concentration was
~100 μM for each peptide. Helium was used as the bath/
buffer gas to improve trapping efficiency and as the collision
gas in the collision cell (ion trap) for collision-induced
dissociation (CID) experiments.
(b) Involvement of Lysine Side Chain Amine in the Water-
Loss Mechanism: Chemical Modification of the Peptide
In order to test the role of the lysine residues in the
water-loss mechanism, we modified the amine functional
groups (N-terminal α-NH2 and side chain ε-NH2 of
lysines) in all the isomeric octapeptides by amine
reductive methylation reaction [39]. The modified
peptides were identified by ESI-MS spectra (see Figure
S2 in the Supporting Information). The peaks at m/z

1970 S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides
b
4
3
2
1
0
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8
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(a) AKAAAKAA
100
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y
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a6
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+
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[M+H]
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7
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356.2
285.1
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7
y
6
200
250
300
350
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450
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550
555.5
200
300
400
500
600
700
m/z
Figure 1. Low energy CID-MS/MS spectra (MS2) of the singly protonated peptide ions [M+H]+ (m/z 700.5) of the peptides (a)
AKAAAKAA-NH2, (b) AAAKKAAA-NH2, (c) KAAAKAAA-NH2, (d) KAAAAAAK-NH2, and (e) AAAKAAAK-NH2. The inset of each
spectrum is the enlarged view of the low m/z range to show all the CID product ions observed. No ion signal was observed
below m/z 200. For convenience the y-ions have been labeled using blue color, b-ions using red color and other ions using
black color. Internal fragments appeared due to the preferential loss of N-terminal lysine from the bn-ions of KAAAAAAK-NH2
are shown by purple arrow in the inset of (d)
784.5 correspond to the singly protonated hexamethy-
lated products of all the peptides. Thus all the primary-
amines in the peptides are converted into the tertiary-
amines upon N,N-dimethylation reaction. The tandem mass

S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides 1971
spectra (MS2) of the singly protonated species of each of
these modified peptides are shown in Figure 2. Unlike in the
case of the unmodified peptides (Figure 1), the bn-ions of the
modified peptides do not undergo any neutral loss of water.
In fact none of the sequence informative fragment ions (e.g.,
an, bn, and yn) of the modified peptides show neutral loss of
water. The most intense peak (base peak) is the b8-ion
corresponding to the loss of NH3 from the protonated C-
terminal amide (Figure 2) analogous to that observed in the
unmodified peptides (Figure 1). This result strongly
b
8
100
(a) AK'AAAK'AA
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b
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c
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8
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(e) AAAK'AAAK'
+
[M'+ H]
784.5
y
5
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7
b
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b
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7
y
4
a
4
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3
a
6
b
7
8
5
5
a
c
4
6
200
300
400
500
600
700
800
m/z
Figure 2. Low energy CID-MS/MS spectra (MS2) of the singly protonated aminomethylated-products [M' + H]+ (m/z 784.5) of
the peptides (a) AKAAAKAA-NH2, (b) AAAKKAAA-NH2, (c) KAAAKAAA-NH2, (d) KAAAAAAK-NH2, and (e) AAAKAAAK-NH2. In
the Figure prime symbol on each lysine residue (K’) indicates that the lysine side chain amine is methylated by two methyl
groups. The N-terminal α-amine is also methylated in each peptide. The inset of the panels (b) and (d) are the enlarged views of
the low m/z range to show all the CID product ions observed. No ion signal was observed below m/z 200. For convenience, the
y-ions have been labeled using blue color, b-ions using red color, and other ions using black color

1972 S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides
suggests that the primary-amine functional groups of the
lysine containing peptides are involved in the loss of water.
In order to determine whether the N-terminal α-NH2
group or the lysine ε-NH2 group is involved in the water-
loss mechanism, we selectively modified this N-terminal α-
NH2 group of the peptides by acetylation [38]. The tandem
mass spectra of all the α-aminoacetyl peptides (see Figures
S3, S4, and S5 for three typical peptides Ac-AKAAAKAA-
NH2, Ac-AAAKKAAA-NH2, and Ac-KAAAKAAA-
NH2, respectively) show that the maximum number of
water loss from the fragment ions is equal to the number of
lysine residue present in that ion analogous to that observed
in case of the unmodified peptides (Figure 1). These results
thus clearly suggest that the lysine side chain amines are
indeed involved in the mechanism to loss the water
molecule from the protonated peptides in the gas phase.
The possible mechanism of this water elimination
could be the acid (H+) catalyzed unimolecular dehydra-
tion reaction between the lysine ε-NH2 group and the
carbonyl group of a suitable amide in the peptide
backbone. This would lead to the formation of a cyclic
imine fused to the peptide backbone (Scheme 1).
According to the mobile proton model [11, 12, 44, 45],
the collisionally activated protonated peptides would
have protonation at all the possible sites (hetero atoms)
including the backbone carbonyls of the amides. The
Scheme 1 shows that the side chain lysine amine acts as
the nucleophile and the carbon center of one of the
carbonyl groups of the peptide backbone acts as an
electrophile on protonation of the amide carbonyl
oxygen. Thus the water eliminated species observed in
the tandem mass spectra of the lysine containing
octapeptides (Figure 1) are possibly cyclic imines fused
to the peptide backbone as proposed in Scheme 1.
imine. The b8-ions were mass selected from the MS2 spectra
(Figure 1) of all the singly protonated isomeric octapeptides
and subjected to further CID to obtain the MS3 spectra
(Figure 3). Figure 3 shows very intense ion abundance for
the species [b8-H2O]+ (m/z 665.5) in the case of the three
peptides AKAAAKAA-NH2, AAAKKAAA-NH2, and
KAAAKAAA-NH2 (Figure 3a–c), unlike in the case of
the peptides KAAAAAAK-NH2, AAAKAAAK-NH2 that
contained a lysine residue at the C-terminal (Figure 3d and
e). The peptides containing C-terminal lysine rather showed
the b7-ions (base peak) as the most abundant species
indicating collisional activation (MS3) causes preferential
loss of a C-terminal neutral lysine, which possibly exists as a
cyclic amide form (ε-caprolactam) [46] (Figure 3d and e).
The water-loss ions [b8 – H2O]+ (m/z 665.5) mass selected
from the MS3 spectra (Figure 3a–c) were subjected to
further CID to obtain the corresponding MS4 spectra
(see Figure S6 in the Supporting Information). The MS4
spectra showed loss of two water molecules in some
product ions (i.e., an – 2H2O, bn – 2H2O, and/or yn –
2H2O ions) that contained two lysine residues, while
only a single water loss was observed for the product
ions (i.e., an-H2O, bn-H2O, and/or yn-H2O ions) that
contained single lysine residue. Thus, the MS4 spectrum
for the peptide AAAKKAAA-NH2 showed no water
loss from b3 and y3-ions (that contained no lysine
residues) but single water loss from b4-, y4-ions (contain
only one lysine residue) and double water loss from
the product ions containing two lysine residues (e.g.,
b5-, b6-, b7-, b8-, or y5-, y6-, y7-ions (see S6b in the
Supporting Information). The observation of the water-
loss ions in the MS4 spectra can be used for mass finger
printing in identifying the amide carbonyl that is
involved in the formation of the cyclic imine (see
Scheme S1 in the supporting information). For example,
the detection of double water loss from b6- and y7-ions
and single water loss from b5- and y6-ions in the MS4
spectrum of the peptide AKAAAKAA-NH2 (see S6a in
the Supporting Information) supports formation of a
seven-membered cyclic imine on water loss from the
lysine containing peptide (see Scheme S1 (B) in the
Supporting Information). These results thus clearly
suggest that the carbonyl carbon of the ith lysine residue
is attacked by the nucleophilic ε-NH2 of the same lysine
residue (i-i cyclization) to form the seven-membered
cyclic imine (see Scheme S1 (B) in the Supporting
Information) fused to the peptide backbone by losing
one water molecule. The simultaneous possibilities of
side reactions involving formation of larger cyclic imines
[eg., by i-(i 1)] cyclization can however not be ruled
out from the present results.
(c) Identification of the Amide Carbonyl Involved in the
Cyclic Imine Formation: Multistage CID Study
Multistage CID experiments (MSn) were carried out to
identify the electrophilic carbonyl carbon that is attacked by
the nucleophile (lysine side chain amine) to form the cyclic
Deletion of Internal Lysine Residue
(a) The Assignment of the Peak at m/z 555.5 in the CID
Scheme 1. A tentative mechanism of the dehydration reac-
tion of the peptide under investigation
Spectra

S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides 1973
555.5
(a) AKAAAKAA
100
214.1
285.1
b 356.2
427.2
484.3
b
−
H O
2
8
4
8
6
4
2
0
80
60
40
20
0
b
8
b
6
b
a
2
683.5
b
8
b
3
b
5
7
200
250
300
350
400
200
250
300
350
400
450
m/z
500
550
600
650
700
−
b
H O
2
(b) AAAKKAAA
8
100
80
60
40
20
0
555.5
427.2
285.1
356.2
484.3
413.3
b
6
b
8
683.5
a
8
b
5
b
7
b
b
3
4
200
250
300
350
400
450
500
550
600
650
700
(c) KAAAKAAA
−
b
8
H O
2
100
80
60
40
20
0
285.1
413.3
484.3
555.5
a
6
b
b
8
683.5
6
a
b
4
y
b
a
8
y
3
4
7
6
a
b
5
b
5
a
4
7
b
2
200
250
300
350
400
450
500
550
b
600
650
700
(d) KAAAAAAK
b
25
20
15
10
5
6
7
100
80
60
40
20
0
214.1
285.1
427.2
356.2
a
a
5
7
b
5
b
2
a
b
6
3
b
b
4
8
0
683.5
200
250
300
350
400
450
500
550
200
250
300
350
400
450
m/z
500
550
b
600
650
700
b
(e) AAAKAAAK
20
15
10
5
6
7
100
80
60
40
20
0
285.1
356.2
427.2
b
5
b
a
a
4
7
6
b
3
b
8
0
150
200
250
300
350
400
450
500
550
683.5
200
250
300
350
400
450
500
550
600
650
700
m/z
Figure 3. CID-MS3 spectra of the b8-ions at m/z 683.5 for the peptides (a) AKAAAKAA-NH2, (b) AAAKKAAA-NH2, (c)
KAAAKAAA-NH2, (d) KAAAAAAK-NH2, and (e) AAAKAAAK-NH2. The inset of the panels (a) (d) and (e) are the enlarged view of
the low m/z range to show all the CID product ions observed. No ion signal was observed below m/z 200. For convenience the
y-ions have been labeled using blue color, b-ions using red color and other ions using black color. The nonsequence ion
fragments, appeared due to the internal lysine deletion, have been shown using green colored vertical arrow
The MS2 spectra of the isomeric octapeptides [M+H]+
charged ion at m/z 555.5 (Figure 1). This ion signal is
(m/z 700.5) showed a peak corresponding to a singly
more intense for the peptides with the C-terminal lysine

1974 S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides
(viz., KAAAAAAK-NH2 and AAAKAAAK-NH2) com-
pared with the other three peptides and corresponds to a
difference in the m/z value of 145 u from the protonated
peptide. This ion signal matches with the expected b7-
ion (Figure 1d and e) produced by the neutral loss of
amidated lysine (mass 145 u) from the C-terminus of
these two peptides (containing C-terminal lysine).
However, this ion signal (m/z 555.5) in the other three
peptides (Figure 1a–c) does not correspond to any direct
sequence ion ( an, bn, xn, yn ) or their neutral loss (water
or ammonia) species. It is interesting to note that this ion
signal is consistently observed in all peptides even in the
MS3 spectra (Figure 3a–e) obtained by CID of the mass
selected b8-ions from the MS2 of the protonated
peptides. The relative ion abundance of this non
sequence ion signal (m/z 555.5) increases from ~2 %–
4 % in the MS2 spectra (Figure 1 a–c) to ~50 %–100 %
in the MS3 spectra (Figure 3a–c) in case of these three
peptides. Moreover, the ion abundance of this peak in the
MS2 spectra (Figure 1 a–c) increases with the normalized
collision energy. The energy resolved CID (MS2) results
(Figure S7 in the Supporting Information) showed that the
fractional abundance of this nonsequence ion (m/z 555.5) is
highest at the normalized collision energy ~45 % and
further increase in the collision energy leads to decrease in
the abundance of this ion.
sequence informative ionsof the m/z 555.5 species, Figure 4
also shows the fragment ions (an-, bn-ions) corresponding
to the CID of the hexapeptide AAAAAA suggesting that
both the lysine residues are deleted from the peptides at the
MS4 stage. Scheme 2 summarizes the gas-phase reactions
described above for a typical peptide AKAAAKAA-NH2.
(c) Mechanism of the Internal Lysine Deletion
Though a simple mechanistic explanation based on the
established fragmentation rules [7] can account for the
neutral loss of terminal lysine (C-/N-terminal) through
formation of bn- or yn ions from the protonated peptide,
the internal lysine loss in the present case cannot be
described by such mechanism.
Recent studies [21, 27–32] have shown that the bn-ions
and their analogues of a peptide may undergo head-to-
tail cyclization, forming a protonated cyclic intermediate
through the nucleophilic attack by the N-terminal α-
amino group on the carbonyl carbon of the C-terminal
oxazolone ring. The authors showed that this cyclic
protonated peptide intermediate [21, 27–32] undergoes
ring opening at different amide bonds to form a series of
the linear oxazolone terminated bn-ions of scrambled
sequences. The cyclization and subsequent ring-opening
process, was thus proposed to lead to scrambling of the
original primary sequence of the bn-ions, which upon
collisional activation could form the daughter ions
corresponding to the neutral loss of an internal residue
from the peptide. Selective deletion of internal lysine by
sequence scrambling following the head-to-tail macro-
cyclization cannot indeed be completely ruled out in the
present case, though no evidence of the deletion of
internal alanine as anticipated from the proposed random
scrambling [21, 27–32] of the peptide sequence was
observed in our results.
In order to assess whether head-to-tail macrocycliza-
tion of the bn-ion (as discussed above) could be a major
pathway for the internal lysine deletion, we selectively
acetylated the N-terminal α-amine of the peptides since
N-acetylation blocks the macrocyclization [28, 47]. It is
interesting to note that the deletion of internal lysine by
collisional activation of the protonated octapeptides as
described above (Figure 1 and Figure 3) indeed takes
place even after the N-acetylation (see Figures S3, S4,
and S5 in the Supporting Information) of the peptides
(m/z 742.4, the singly protonated N-acetylated peptides).
Analogous to that observed for the unmodified peptides,
the results in the tandem mass spectra (Figures S3, S4,
and S5 in the Supporting Information) show that an ion
signal ( m/z 597.4 ) corresponding to the loss of 145 u
(residue mass of Lys + mass of NH3) from the
protonated N-acetylated peptides suggesting deletion of
internal lysine residue. This indicates that the deletion of
internal lysine as observed in the present case may also
occur in a different pathway other than the sequence
scrambling fragmentation pathway [21, 27–32].
(b) Possible Structure of the Ion Fragment at m/z 555.5:
Multistage CID study
As discussed above, the singly charged ion of m/z 555.5,
formed by loss of 145 u from the protonated peptide
[M+H]+ ion possibly corresponds to the loss an amidated
lysine or to the simultaneous loss of a lysine residue and
an ammonia molecule. Figure 1d and e suggests that this
ion could be formed by the loss of the terminal amidated
lysine from the peptides KAAAAAAK-NH2 and AAA-
KAAAK-NH2 and corresponds to the direct sequence
b7-ion in these two peptides. However, the non sequence
ion at m/z 555.5 observed in case of the other three peptides
(Figure 1 a–c) cannot be directly formed by loss of the
terminal residue, rather the simultaneous loss of an N-
terminal or an internal lysine (residue mass 128 u) and an
ammonia molecule (17 u) may give rise to this species. In
order to ascertain the origin of this non sequence ion, CID
studies of the mass selected ions (m/z 555.5) from the MS3
spectra (Figure 3a–c) were carried out (Figure 4).
The possible sequences of the ion at m/z 555.5
produced from each of the octapeptides were derived
from the analyses of the mass finger prints in the MS4
spectra (Figure 4). The results in Figure 4a–c suggest
that this ion is produced by the loss of any one of the two
lysine residues either from the inside or from the N-
terminal of the peptide along with a neutral loss of
ammonia from the C-terminal amide. This also may be
produced by the loss of one lysine residue from the
corresponding b8 -ions of the peptides. Apart from the

S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides 1975
H O loss
2
537.5
AKAAAAA
100
(a)
AAAAAA
AAAAKAA
80
60
b
b
5
6
b
2
40
20
0
a
2
a
b
5
6
4
a
555.5
550
b
3
150
200
250
300
350
400
450
500
b
5
100
80
60
40
20
0
AAAKAAA
AAAAAA
(b)
H O loss
2
537.5
b
4
b
b
5
6
a
4
b
4
b
a
7
6
b
6
a
5
555.5
150
200
250
300
350
400
450
500
550
H O loss
2
537.5
b
/
(c) AAAKAAA
5
5
100
80
60
40
20
0
AAAAAA
KAAAAAA
a
4
b
4
b
a
b
b
3
6
5
b
a
a
2
5
6
a
2
555.5
150
200
250
300
350
400
450
500
550
−
b
H O
2
7
100
80
60
40
20
0
KAAAAAA
AAAAAA
(d)
a
5
a
b
5
b
5
b
5
a
4
6
a
7
b
7
b
a
6
6
b
4
b
2
b
a
555.5
b
4
6
3
b
3
150
200
250
300
350
400
450
500
550
b
5
100
80
60
40
20
0
AAAKAAA
AAAAAA
(e)
b
b
7
555.5
4
b
b
a
6
b
a
6
a
a
6
a
4
5
7
b
5
4
4
150
200
250
300
350
400
450
500
550
m/z
Figure 4. CID-MS4 spectra of the fragment ions at m/z 555.5 from the peptides (a) AKAAAKAA-NH2, (b) AAAKKAAA-NH2, (c)
KAAAKAAA-NH2, (d) KAAAAAAK-NH2, and (e) AAAKAAAK-NH2 under the same collision energy (28 %). No ion signal was
observed below m/z 150. In each panel of the figure, different colors have been used to label the fragment ions of the same
colored amino acid sequence shown in top left corner of each panel
Furthermore, collisional activation (MS2) of the amino-
methylated peptides (m/z 784.5) shown in Figure 2a–c did
not show any ion signal at m/z 611.5 corresponding to the
deletion of an internal lysine (methylated). The amino-
methylated octapeptides containing a lysine residue at the C-
terminal however showed the direct sequence ion (b7-ion) at
m/z 611.5 (Figure 2d and e) corresponding to the deletion of
the C-terminal ε-aminomethylated lysine amide (mass 173).
These results suggest that the internal lysine deletion from
the isometric octapeptides might be a consequence of the

1976 S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides
Me
MeO
H
AKAAAK
NH CH C N C
C NH
3
H
O
[AKAAAKAA-NH2 +H]+
m/z 700.5
NH3 loss
NH2
Me
O
C
O
HN
H
H
H
C
AAAKA
N C
H
N
O
O
Me
b8 -ion in form of oxazolone structure
m/z 683.5
[AKAAAKAAox +H]+
Proton Transfer
NH2
HN
NH
OH
2 OH
Scheme 2. Schematic representation of the internal lysine loss
from the gaseous protonated peptide (C-terminal amidated) in
the tandem mass spectrometric (MSn) experiments
AAox
AAox
N
N
H
H
HN
AAAKA
AAAKA
O
O
water-loss phenomenon discussed in previous section, and
indeed involves the free ε-NH2 of the lysine residue.
[AKAAAKAAox +H]+
A
Path I
-H2N-AAox
Path II
-H2O
Based on the above discussion, a mechanism involving
formation of a cyclic imine intermediate leading to the
deletion of the internal lysine from the collisionally
activated protonated octapeptides has been proposed as
shown in Scheme 3 (for a typical peptide AKAAAKAA-
NH2). Such seven member cyclic imine involving the
lysine residue has been suggested to be formed by water
loss from the peptide ion (Scheme S1B in the Supporting
Information). Scheme 3 shows that the b8-ion may exist in
an oxazolone [15] structure with the charge (proton)
delocalized on the whole peptide ion [11, 45]. Protonation
of the carbonyl oxygen of a lysine residue would enhance
the electrophilicity of the amide carbon and allow
formation of the seven membered cyclic intermediate A
on nucleophilic attack of the amide carbon by the free ε-
NH2 of the same lysine residue. The formation of
analogous cyclic intermediates of larger size cannot also
be ruled out, but such intermediates would not result in
deletion of the internal lysine as shown in Scheme 3.
Scheme 3 shows that the intermediate A can undergo
cleavage in two possible pathways: Path I forms a bn-ion
(b6-ion, B in the typical peptide shown in Scheme 3), which
contains a protonated cyclic amide (ε-caprolactam) formed
by the loss of a neutral peptide fragment (AAox in
Scheme 3, ‘ox’ indicates C-terminal oxazolone form) from
the C-terminus in the intermediate A. The protonated cyclic
amide (ε-caprolactam) has been shown to be more stable
than the corresponding isomeric oxazolone structure [46,
48] of the bn-ion containing lysine at the C-terminus. The
other competitive pathway (path II) forms a cyclic imine
intermediate (C) by the elimination of one H2O from the
intermediate A. This cyclic imine intermediate (C) is the
water-loss product of b8-ion (b8-H2O ion) as described in
the previous section (neutral loss in Scheme S1 in the
NH
NH2
H
N
AAox
AAAKA
O
AAAKA
O
HN
HN
O
C
B
b6-ion at m/z 541.3 b8-H2O ion at m/z 665.5
Proton Transfer
H
N
N
[AKAAAAAox +H]+
m/z 555.5
NH AAox
AAAKA
H
HN
NH
D
E
O
C
Scheme 3. The proposed mechanism of the deletion of the
internal lysine residue from the gaseous protonated peptide
upon collisional activation
Supporting Information). Scheme 3 shows that the species
C is the precursor for the loss of internal lysine residue that
is involved in the formation of the imine ring. A
unimolecular bond rearrangement (Scheme 3) in the
species C would lead to deletion of the neutral cyclic
imine D (3,4,5,6-tetrahydro-2 H-azepin-6-imine, M.W.
110 ) forming the lysine deleted protonated peptide
(species E). This scheme is further supported by the
observation that the collisional activation of the species C
(b8-H2O ion, m/z 665.5) showed deletion of the cyclic
imine (species D, M.W. 110) corresponding to loss of an
internal lysine, and a neutral loss of water from the second
lysine residue resulting in an ion at 537.5 (species E-H2O,
see Figure S6 in the Supporting Information). The resulting
peptide fragment (E-H2O) is similar to the species C (with

S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides 1977
one lysine less than that in species C) and would
subsequently release another cyclic imine (species D)
leading to the deletion of the second lysine residue to form
the protonated hexapeptide ion ([AAAAAAox + H]+). It is
important to note that the species B (b6-ion, m/z 541.3), C
(b8-H2O ion, m/z 665.5), and E (m/z 555.5) shown in
Scheme 3 have been detected and characterized by tandem
mass spectrometry but the species D, being neutral, could
not be detected. The formation of a bn-ion analogous to
species A was also proposed earlier [46, 48] to be involved
in the formation of ε-caprolactam terminated peptide
fragment ion (analogous to species B) as shown in Path I
of Scheme 3.
ion signals at m/z 555.5 and m/z 563.5 (see the inset of
Figure 5a) corresponding to the loss of K* and K
respectively from the b8-ion of the labeled peptide. Further-
more, analogous to that observed in case of the unlabeled
peptide (Figure 3), the MS3 on the b8-ion of the labeled
peptide resulted in enhanced loss of both K* and K leading
to increase in the relative abundance of both the ion signals
at m/z 555.5 and m/z 563.5 (see Figure 5b). The tandem
mass analyses (MS4) showed that the ion signal at m/z 555.5
corresponds to the sequence AKAAAAAox (Figure S8a in
the Supporting Information), while the ion signal at m/z
563.5 corresponds to the sequence AAAAK*AAox (Figure
S8b in the Supporting Information). Moreover, collisional
activation (MS4) of both these ions produced the peptide
fragment AAAAAAox by deletion of the remaining lysine
residue as observed in case of the unlabeled analogues
(Figure 4). These results unambiguiously support that
collisional activation of the protonated octapeptide indeed
leads to deletion of the internal lysine residue as proposed in
the Scheme 3.
ESI-MS of Isotopically Labeled Octapeptides
In order to achieve further insight into the gas-phase
chemistry of the isomeric protonated octapeptides, one of
the lysine residues of the peptide was labeled by 15N and
13C isotopes. The electrospray ionization mass spectra of the
isomeric octapeptide containing isotopically labeled lysine
residue produced singly protonated species [M*+H]+ at m/z
708.5 and doubly protonated species [M*+2 H]+ at m/z
354.8 in the gas phase. Analogous to the unlabeled peptide
(Figure 1), collisional activation (MS/MS) of the singly
protonated isotopic peptide [M*+H]+ (m/z 708.5) produced
the characteristic bn and yn ions. Figure 5a shows the MS2
spectrum of the isotopically labeled peptide AKAAAK*
AA – NH2 (K* represents labeled lysine), which shows the
Redesigning New Peptides to Get a General
Insight of the Lysine Deletion Phenomena
So far we have discussed the gas-phase fragmentation chemistry
of the positional isomers of the peptide containing six alanine
and two lysine residues. In order to assess whether the deletion
of the internal lysine by collisional activation of the protonated
peptide indeed applies to any lysine containing peptide or not,
555.5
b
3.0
2.5
2.0
1.5
1.0
0.5
0.0
AKAAAK*AA
8
100
80
60
40
20
0
(a)
563.5
b
b
7
6
+
y
[M+H]
708.5
6
555 558 561 564
m/z
b
5
200
300
400
500
600
700
m/z
555.5
(b)
b
−
H O
2
8
AKAAAK*AA
100
80
60
40
20
0
484.3
356.2
563.5
b
b
8
6
a
691.5
8
b
b
b
4
3
5
b
7
200
300
400
500
600
700
m/z
Figure 5. Low energy CID (a) MS2 spectrum of the singly protonated peptide AKAAAK*AA-NH2 (m/z 708.5), (b) MS3 spectrum
of the b8-ion, (c) MS4 spectrum of the fragment ion at m/z 555.5, and (d) MS4 spectrum of the fragment ion at m/z 563.5. The K*
in the peptide sequence indicates that this amino acid (lysine) is isotopically labeled (13C & 15N). The inset of (a) is the enlarged
view of the m/z range from 553 to 565. For convenience the y-ions have been labeled using green color, b-ions using red color,
and other ions using black color in (a) and (b). Apart from the ions at m/z 555.5 and 563.5 the other nonsequence ion fragments,
appeared due to the internal lysine deletion, have been shown using violet colored vertical arrow in (b)

1978 S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides
we investigated the gas-phase fragmentation chemistry of a new
peptide of sequence YAKGFKL-NH2. The amino acids
containing carboxylic acid side chain (glutamic acid or aspartic
acid) or guanidium side chain (arginine) were avoided in
designing the peptide (YAKGFKL-NH2) as they have earlier
been shown to introduce additional complexity in the tandem
mass spectra [49].
analogous to that observed in case of the isomeric
octapeptides (Figure 4). These results thus suggest that the
deletion of the internal lysine residue from the protonated
peptide possibly does not depend on the sequence of the
peptide.
Lysine Deletion from Multiply Charged Peptides
The MS/MS spectrum (Figure 6a) of the singly proton-
ated heptapeptide YAKGFKL-NH2 (C-terminal amidated,
m/z 825.5) shows direct sequence ions (an, bn, and yn-ions)
along with their water-loss products. The number of water
loss was directly correlated with the number of lysine
residues in the fragment ions of this peptide, as observed
in case of the isomeric octapeptides. The MS2 spectrum
(Figure 6a) of the peptide also shows an intense ion signal at
m/z 680.3 (25 % relative abundance in MS2) indicating the
loss of ~145 u corresponding to the loss of a lysine residue
and an ammonia molecule. This suggests that the deletion of
internal lysine takes place from the b7-ion of this heptapep-
tide. The tandem mass analyses (MS3) suggested (Figure 6b)
that the ionic species of m/z 680.3 indeed correspond to the
loss of an internal lysine residue from the protonated
heptapeptide, and have the sequence YAKGFLox or YAGF-
KLox (ox refers to oxazolone). These fragment ions (m/z
680.3) undergo further deletion of the remaining lysine
residue to generate YAGFLox peptide fragment (Figure 6b)
The doubly protonated peptide at m/z 350.8 observed in the
mass spectra (Figure S1) of the isometric octapeptides, was
mass selected and subjected to collision-induced dissocia-
tion. The MS2 spectra of the doubly protonated peptides
(Figure S9 in the Supporting Information) show that the
attachment of two protons provides dissociation pathways
producing both singly charged as well as doubly charged
fragment ions. All those spectra showed the appearance of a
singly positive and a doubly positive ion respectively at m/z
555.5 and m/z 278.3 corresponding to the loss of an internal
lysine residue followed by a neutral loss (NH3) from the
peptides. The MS2 of the doubly protonated heptapeptide
(YAKGFKL-NH2) also showed internal lysine deletion
(Figure S10 in the Supporting Information) analogous to
that observed in case of the singly protonated peptide
(Figure 6). Thus, the deletion of the internal lysine residue
is also possible by collisional activation of the multiply
charged peptides.
(a)
(b)
Figure 6. (a) Low energy CID-MS/MS (MS2) spectrum of the singly protonated peptide YAKGFKL-NH2 (m/z 825.5). For
convenience, the y-ions have been labeled using blue color, b-ions using red color and other ions using black color. (b) CID-
MS3 spectrum of the ion at m/z 680.3 derived from the MS2 of the same peptide. Here different colors have been used to label
the fragment ions of the same colored amino acid sequence shown in top left corner. No ion signal was observed below m/z
200

S. Banerjee and S. Mazumdar: Deletion of the Internal Lysine Residue from Gaseous Protonated Peptides 1979
5. Sleno, L., Volmer, D.A.: Ion Activation Methods for Tandem Mass
Spectrometry. J. Mass Spectrom. 39(10), 1091–1112 (2004)
6. McLuckey, S.A.: Principles of Collisional Activation in Analytical
Mass Spectrometry. J. Am. Soc. Mass Spectrom. 3(6), 599–614 (1992)
Conclusions
The gas-phase collision-induced dissociation of a series of
lysine containing synthetic peptides showed water-loss (neutral
7. Banerjee, S.; Mazumdar, S.: Electrospray Ionization Mass Spectrome-
loss) from the sequence informative ions (an, bn, and yn-ions).
The number of water-loss was found to be equal to the number
of lysine residues present in the corresponding fragment ions.
The tandem mass spectrometry of the peptides, their amino-
methylated derivatives, and the N-terminal acetylated deriva-
tives showed that the gas-phase reaction between the side-chain
amine of a lysine residue and the carbonyl group of the same
residue results in the formation of a seven-membered cyclic
imine fused to the backbone of the peptide fragment is involved
in the water-loss mechanism.
As a consequence of this water-loss phenomenon, the
peptide ion undergoes deletion of the internal lysine residue
upon collisional activation by intramolecular bond rear-
rangement. The detailed mechanistic studies suggested that
the deletion of the internal lysine takes place by a gas-phase
degradation reaction of the cyclic imine containing peptide
ion intermediate through releasing the corresponding cyclic
imine from the interior of the peptide. The results have been
confirmed by using isotopically labeled lysine residues and
also by redesigning new peptide sequence, which suggest
that the gas-phase deletion of the internal lysine on
collisional activation of the peptide is possibly independent
of the nature of the other residues in the peptide. This
phenomenon may thus potentially have immense application
in designing new proteomic methods for unambiguous
identification of lysine residues in a protein or a peptide by
mass spectrometry and further studies are in progress in our
laboratory in this direction.
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Acknowledgment
The authors thank Mr. Bharat T. Kansara for his help.
Thanks also to Mr. C. Muralidharan and Mr. Bidyut Sarkar
for their help during peptide synthesis. This work was
supported by Tata Institute of Fundamental Research.
Supporting Information Available.
Breakdown graphs, mass spectra of the peptides and their
derivatives, and schematic representation of the peptide dehy-
dration reactions are shown in the Supporting Information.
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