The ornithine effect in peptide cation dissociation

Article abstract of DOI:10.1002/jms.3233

Facile cleavage C-terminal to ornithine residues in gas phase peptides has been observed and termed the ornithine effect. Peptides containing internal or C-terminal ornithine residues, which are formed from deguanidination of arginine in solution, were fragmented to produce either a y-ion or water loss, respectively, and the complementary b-ion. The fragmentation patterns of several peptides containing arginine were compared to those of the ornithine analogues. Conversion of arginine to ornithine results in a decrease of the gas phase proton affinity of the residue, thereby increasing the mobility of the ionizing proton. This alteration allows the nucleophilic amine to facilitate a neighboring group reaction to induce a cleavage of the adjacent amide bond. The selective cleavage at the ornithine residue is proposed to result from the highly favorable generation of a six-membered lactam ring. The ornithine effect was compared with the well-known proline and aspartic acid effects in peptide fragmentation using angiotensin II, DRVYIHPF and the ornithine analogue, DOVYIHPF. Under conditions favorable to either the aspartic acid (i.e. singly protonated peptide) or proline effect (i.e. doubly protonated peptide), the ornithine effect was consistently observed to be the more favorable fragmentation pathway. The highly selective nature of the ornithine effect opens up the possibility for conversion of arginine to ornithine residues to induce selective cleavages in polypeptide ions. Such an approach may complement strategies that seek to generate non-selective cleavages of the related peptides. Copyright 2013 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/jms.3233

Research article
Received: 9 March 2013
Revised: 6 May 2013
Accepted: 7 May 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3233
The ornithine effect in peptide
cation dissociation
William M. McGee and Scott A. McLuckey*
Facile cleavage C-terminal to ornithine residues in gas phase peptides has been observed and termed the ornithine effect.
Peptides containing internal or C-terminal ornithine residues, which are formed from deguanidination of arginine in solution,
were fragmented to produce either a y-ion or water loss, respectively, and the complementary b-ion. The fragmentation
patterns of several peptides containing arginine were compared to those of the ornithine analogues. Conversion of arginine
to ornithine results in a decrease of the gas phase proton affinity of the residue, thereby increasing the mobility of the ionizing
proton. This alteration allows the nucleophilic amine to facilitate a neighboring group reaction to induce a cleavage of the
adjacent amide bond. The selective cleavage at the ornithine residue is proposed to result from the highly favorable generation
of a six-membered lactam ring. The ornithine effect was compared with the well-known proline and aspartic acid effects in
peptide fragmentation using angiotensin II, DRVYIHPF and the ornithine analogue, DOVYIHPF. Under conditions favorable
to either the aspartic acid (i.e. singly protonated peptide) or proline effect (i.e. doubly protonated peptide), the ornithine effect
was consistently observed to be the more favorable fragmentation pathway. The highly selective nature of the ornithine effect
opens up the possibility for conversion of arginine to ornithine residues to induce selective cleavages in polypeptide ions. Such
an approach may complement strategies that seek to generate non-selective cleavages of the related peptides. Copyright ©
2013 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: selective cleavage; ornithine; lactam formation; deguanidination
side-chain effects). There are currently many examples of residues
Introduction
that exhibit selective cleavages under specific conditions.[22–33]
Cleavages observed at aspartic acid[23,26–28] and proline[22,23,25,31]
Mass spectrometry has emerged as the leading tool for identifying
both proteins and peptides. With the advent of soft ionization
methods such as matrix-assisted laser desorption ionization
(MALDI)[1] and electrospray ionization (ESI),[2,3] large molecules,
particularly biomolecules, can be readily ionized without inducing
fragmentation thereby facilitating mass determination. Primary
structure determination is usually approached by fragmenting
ions along the peptide backbone. For protonated proteins and
peptides, activation of the ions via collisions or absorption of IR
photons results most commonly in backbone fragmentation at
the amide bonds. Such behavior is common to all dissociation
methods that give rise to vibrationally excited even-electron
peptide and protein cations, such as, for example, collision-
induced dissociation (CID),[4–6] surface-induced dissociation[7–9]
and infrared multiphoton dissociation.[10–13]
residues are among the most commonly observed of these facile
cleavages. These cleavages are related to the mobile proton
effect, yet the individual processes are quite different. For
the aspartic acid effect, if a proton is sequestered away from
the aspartic acid residue, and therefore not mobile, cleavage
C-terminal to the aspartic acid is observed by cyclization of the
side chain to produce a terminal succinic anhydride and a new
primary amine. This type of cleavage that does not directly involve
the ionizing proton is sometimes described as a charge-remote
fragmentation pathway and is readily observed with a nearby
arginine residue, which serves to sequester the proton away from
aspartic acid. Alternatively, if there is a mobile proton, the amide
bond of proline has a higher proton affinity than neighboring
amide bonds, resulting in a highly favorable cleavage N-terminal
to the proline due to the higher basicity of the tertiary nitrogen.
In this scenario, the ionizing proton is directly involved in the
cleavage and is therefore said to be a charge-directed fragmentation
pathway. Both of these cleavage phenomena have been found
to dominate spectra under conditions favorable to the
particular pathways.
The mobile proton model[14–20] is widely accepted as rationalizing
the fragmentation behavior of protonated peptides in the gas
phase. Basic sites, such as the N-terminal amine, and the side
chains of arginine, lysine and histidine are generally accepted
to be the locations for excess protons in ions of low internal
energy. Activation of the peptide ion increases the mobility of
the proton,[16,19] allowing it to populate different sites along the
peptide backbone. When a backbone amide bond is protonated,
cleavage is facilitated to produce b- and y-type ions.[21] Although
cleavage at other bonds in the peptide backbone may occur, this
is the most common cleavage observed for CID experiments.
In several cases, some of these backbone cleavages are found
to be much more favorable than others, depending on the
amino acid composition of the peptide (i.e. there can be strong
*
Correspondence to: S. A. McLuckey, 560 Oval Drive, Department of
Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA. E-mail:
mcluckey@purdue.edu
Department of Chemistry, Purdue University, West Lafayette, IN, 47907-2084, USA
J. Mass Spectrom. 2013, 48, 856–861
Copyright © 2013 John Wiley & Sons, Ltd.

The ornithine effect: selective cleavages observed in ornithinated peptides
Ornithine is an amino acid that is not coded by DNA but is
produced in nature via deguanidination of arginine and plays an
important role in the urea cycle. Chemical deguanidination of
arginine in a polypeptide results in the conversion of a guanidine
containing side-chain to a d-amine-containing side chain. The
proton affinity of ornithine, the side chain of which is one
methylene group shorter than lysine, is therefore expected to be
very similar to that of lysine and much less than that of
arginine.[34,35] The conversion of arginine to provide enhanced
sequence coverage has been explored previously.[36–38] Conversion
of arginine to an ornithine residue in a polypeptide, therefore,
might be expected to alter the fragmentation pattern of an ion
derived from the peptide simply due to more facile proton mobility
in the ornithine case, much like the difference observed between
the same peptide with a lysine exchanged for an arginine.
However, the conversion of arginine to ornithine has a much
greater effect than does the substitution of a lysine for an
arginine. Specifically, the introduction of ornithine exhibits a
classic case of a neighboring group effect,[39] which leads to a
highly selective cleavage C-terminal to the ornithine residue. The
conversion of arginine to ornithine residues within the context
of the tandem mass spectrometry (MALDI-post-source decay) of
N-terminal tris(2,4,6-trimethoxyphenyl)phosphonium derivatization
has been reported,[38] but the effect described here, although
observed as a minor process, was not discussed. In this report,
the ornithine effect is described and compared with the aspartic
acid and proline effects using a peptide in which all three
processes can compete. We have found that the ornithine effect
appears to dominate. The conversion of arginine to ornithine in
peptide and protein ions may therefore be useful when it is
desirable to introduce a highly selective decomposition route.
Scheme 1. Conversion of arginine to ornithine.
Concord, ON, Canada), modified with a home-built, alternately
pulsed nanoelectrospray ionization (nESI) source.[41] Following
positive mode nESI, cations were transferred to Q3 where they
were mass selected. Following isolation, the ions were activated
and subsequently fragmented via ion trap CID. Ions were mass
analyzed via mass-selective axial ejection.[42]
Results and discussion
The ornithine effect
A comparison of the fragmentation patterns of arginine- versus
ornithine-containing peptides is shown in Fig. 1, with the peptide
AAAAAXA, where X is either R or O. Figure 1(a) shows CID of
[AAAAARA + H]+, which produces several sequence ions as well
as many neutral losses. In the case of Fig. 1(b), [AAAAAOA + H]+
is cleaved C-terminal to ornithine, forming two fragments, b6
and b5. As the N-terminal amine of the b-ion is the only basic site
remaining, the proton is assumed to be bound there. However, if
there was a basic residue C-terminal to ornithine, one would
expect to see a large abundance of the corresponding y-ion.
It should be noted that although it is commonly accepted that
b-ions are terminated in cycles,[21] as either a diketopiperazine
or an oxazalone, the b-ions formed from the ornithine effect
are terminated with a lactam.
Experimental section
Materials
The peptide AAAAARA was custom synthesized from NeoBioSci
(Cambridge, MA). Angiotensin II human (DRVYIHPF) and hydrazine
hydrate were purchased from Sigma Aldrich (St. Louis, MO). The
peptides YGGFLR and YGGFLK were purchased from CPC Scientific
(Sunnyvale, CA). The peptides GAILPGAILR and GAILDGAILR were
purchased from SynPep (Dublin, CA).
This dramatic difference between the spectra of Fig. 1 is proposed
to arise from a facile new fragmentation channel open to ornithine-
Arginine hydrazinolysis
Aqueous peptide solutions were prepared to concentrations up to
approximately 100 mM. The procedure used has been modified
slightly from previous reactions.[38,40] 50 ml of hydrazine hydrate
was added to 50 ml of the peptide solution in a 1.5 ml vessel. The
reaction mixture was then mixed briefly and allowed to react in
a 60 ꢀC water bath for periods between 2 and 16 h. Complete
hydrazinolysis was observed for the longer time, while a mixture
of modified and unmodified peptides was observed after 2 h of
reaction. The deguanidination reaction is illustrated in Scheme 1.
For peptides containing additional arginine residues, it has been
found to be beneficial, but not necessary, to use a slightly greater
amount of hydrazine.
Figure 1. Comparison of the fragmentation patterns between arginine-
and ornithine-containing peptides. (a) CID of [AAAAARA + H]+ produces
several sequence fragments, neutral losses, and unidentifiable peaks.
(b) CID of [AAAAAOA+ H]+ produces a major cleavage C-terminal to
ornithine. Ammonia loss is labeled with an asterisk (*), water loss is labeled
with a degree sign (ꢀ). Precursor ion fragmented (M) is denoted with a
lightning bolt ( ).
Mass spectrometry
Mass spectrometry experiments were performed on QqQ tandem
mass spectrometers (QTRAP 2000 and QTRAP 4000, AB Sciex,
J. Mass Spectrom. 2013, 48, 856–861
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W. M. McGee and S. A. McLuckey
Scheme 2. (Left) Nucleophilic attack of the carbonyl carbon by the
amine results in the formation of (Right) a six-membered lactam and
cleavage at the C-terminal carbonyl carbon bond of ornithine. If ornithine
is N-terminal or internal, HR is an amine (HNH-R); if ornithine is the
C-terminal residue, HR is water loss (H2O).
containing peptides. An initial nucleophilic attack of the carbonyl
carbon by the g-amine of ornithine, resulting in cyclization and the
formation of a six-membered lactam, gives rise to the abundant b6
ion. This mechanism gives rise to what is being termed the ‘ornithine
effect’, as illustrated in Scheme 2. For N-terminal or internal ornithine
residues, the R in Scheme 2 is an amide nitrogen, such that HR
results in the formation of a primary amine (H2N-peptide). For
C-terminal ornithine residues, the R in Scheme 2 is an OH, such
that HR is water (H2O). It should be noted that the ionizing proton
is not shown in the mechanism of Scheme 2 as this process
involves the nucleophilic attack from an unprotonated amine. This
has been tested by acylating the N-terminal amine of AAAAAOA
with a fixed charge, trimethyammoniumbutyrate (TMAB), to form
the cationic but unprotonated peptide [TMAB-AAAAAOA]+.
Activation of this peptide resulted in a highly selective cleavage
C-terminal to the ornithine, producing a b6 ion (Fig. S-1).
Figure 2. Comparisons of CID behavior of small peptides with basic
C-terminal residues. (a) CID of [YGGFLR + H]+; (b) CID of [YGGFLK + H]+;
(c) CID of [YGGFLO + H]+. Ammonia loss is labeled with an asterisk (*),
water loss is labeled with a degree sign (ꢀ). Precursor ion fragmented
(M) is denoted with a lightning bolt ( ).
result from the formation of a lactam. The enhanced loss of water
from ornithine has been observed previously[43–45] and has been
ascribed to ring size effects. CID of the water loss product in Fig. 2(c)
produces almost entirely the intact b5 ion (see Fig. S-4(d)). This
result is fully consistent with the hypothesis that the loss of H2O
is from the C-terminal lactam formation and that subsequent
fragmentation leads to loss of 3-amino-2-piperidinone to generate
a b5 ion. When the protonated YGGFLO ion was subjected to a
higher activation amplitude, the same products observed in the
CID of the water loss ion (Fig. S-4(d)) were observed (see Fig. S-4(c)),
and no additional products were seen, which suggests that the
greater degree of fragmentation at higher activation amplitudes
arises largely from sequential decomposition following water loss.
This hypothesis was further tested by esterifying the carboxy
group of YGGFLO, such that activation of YGGFLO-OMe should
produce a methanol loss while any water loss observed would
be from an alternate fragmentation pathway.[46,47] Activation of
[YGGFLO-OMe + H]+ was found to produce almost exclusively loss
of methanol from the precursor, with a much smaller loss of water
observed as well (Fig. S-5). We note that protonated YGGFLK
should also be able to undergo an analogous process with the
C-terminal lysine generating a seven-membered lactam ring.[29]
This may very well occur, as there is significant water loss, and
many of the products could result from sequential fragmentation
from the water loss product. However, there are also several
relatively abundant fragments in the data for protonated YGGFLK,
such as the y4 and y5 ions, which cannot arise from the water loss
process. Analogous ions are notably absent in the protonated
YGGFLO data (see Fig. S-4(c)). Apparently, the ability to form the
six-membered lactam makes the reaction of Scheme 2 significantly
more competitive than the analogous reaction with protonated
YGGFLK to form the seven-membered lactam.
Subsequent fragmentation of the b6 ion in Fig. 1(b) via an MS3
experiment produced almost exclusively the b5 ion (Fig. S-2(d)),
which suggests that the b5 ion in Fig. 1(b) could arise largely
or exclusively via sequential fragmentation. Collisional activation
of the [AAAAAOA + H]+ precursor at a higher amplitude than
was used to generate Fig. 1(b) gave rise to an abundant b5 ion
(Fig. S-2(c)), which is consistent with sequential fragmentation via
the b6 ion. Similar fragmentation behavior of the lactam-terminated
bn ion to produce the bnÀ1 has been observed in lysine-
containing peptides by Kish and Wesdemiotis.[29] For lactam-
terminated bn ions, fragmentation produces both the bnÀ1 ion
and 3-amino-2-piperidinone(Fig. S-3).
Peptides containing a c-terminal ornithine
A simple example of a case in which ornithine is the C-terminal
residue is provided in Fig. 2, which compares the CID behavior of
YGGFLX, where X is either R (Fig. 2(a)), K (Fig. 2(b)) or O (Fig. 2(c)).
Lysine- and arginine-terminated peptides are generally produced
from tryptic digestion of proteins, while the ornithine-terminated
peptide would be produced from deguanidination of the arginine-
terminated peptide. The behavior of a C-terminal ornithine in a
peptide is therefore relevant for a strategy that would involve
the conversion of arginine to ornithine in tryptic peptides.
Figures 2(a) and 2(b) both show extensive backbone cleavage
with a somewhat higher degree of spectral complexity, as
reflected in the appearance of more low abundance fragments,
observed in Fig. 2(a) from protonated YGGFLR. The latter observation
is likely due to the somewhat higher activation amplitude required
to generate a similar extent of precursor ion depletion with
protonated YGGFLR than for protonated YGGFLK. The greater
kinetic stability of arginine protonated peptides is fully consistent
with the mobile proton model.[16] Figure 2(c) shows almost
exclusive loss of water from the peptide, which is presumed to
It is of interest to examine the C-terminal ornithine effect
summarized above in peptides where the proline or aspartic acid
effects can also play roles. The model tryptic peptides GAILPGAILX
and GAILDGAILX, where X = R or O, were therefore investigated.
The proline effect is considered for singly protonated peptides
[GAILPGAILR + H]+ and [GAILPGAILO + H]+ in Fig. 3. A mobile
proton is regarded to be a prerequisite for the observation of a
dominant proline effect (i.e. cleavage at the N-terminal side of
proline).[22,23,25,31] In the case of [GAILPGAILR + H]+, the proton is
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The ornithine effect: selective cleavages observed in ornithinated peptides
Figure 3. Comparisons of the proline effect between arginine- and
ornithine-containing peptides. (a) CID of [GAILPGAILR + H]+; (b) CID of
[GAILPGAILO + H]+; (c) CID of [GAILPGAILO + H-H2O]+. Ammonia loss is
labeled with an asterisk (*), water loss is labeled with a degree sign (ꢀ).
Precursor ion fragmented (M) is denoted with a lightning bolt ( ).
Figure 4. Comparisons of the aspartic acid effect with arginine- and
ornithine-containing peptides. (a) CID of [GAILDGAILR+ H]+; (b) 25Â zoom
in of CID of [GAILDGAILR + H]+ from top; (c) CID of [GAILDGAILO + H]+.
Ammonia loss is labeled with an asterisk (*), water loss is labeled with a
degree sign (ꢀ). Precursor ion fragmented (M) is denoted with a lightning
bolt ( ).
effectively sequestered on the arginine residue, thereby significantly
reducing the preferential N-terminal cleavage pathway. The product
ion spectrum from CID of this peptide ion is shown in Fig. 3(a). A
variety of products are observed that includes sequence ions
and internal cleavages. Products expected from a proline effect
(i.e. y6 and b4 ions) are observed but are not particularly dominant.
Conversion of arginine to ornithine is expected to enhance proton
mobility, and, as expected, this modification substantially changes
the fragmentation spectrum, as observed in Fig. 3(b) for CID of
[GAILPGAILO + H]+. Evidence for a more pronounced proline
effect is reflected in the abundant y6-H2O product ion, which is
indicated in the figure as yo6. Interestingly, only a very small y6 ion
is observed. Although the loss of water after formation of the y6
ion cannot be precluded, CID of the H2O loss of [GAILPGAILO + H]+
(Fig. 3(c)) produces a nearly identical spectrum to that of CID of
intact [GAILPGAILO + H]+ (Fig. 3(b)). The only significant difference
is that there are no complete y-ions, only dehydrated y-ions, in the
case of dehydrated [GAILPGAILO + H]+. The similarity between the
two spectra is consistent with the interpretation that most of
the product ions in Fig. 3(b) arise from sequential fragmentation
following water loss from the precursor ion with concomitant
lactam formation (Scheme 2). At lower activation amplitude,
water loss dominates the product ion spectrum of [GAILPGAILO + H]+
(Fig. S-6(b)), which further supports the proposed sequence of
events. In this scenario, the prominent b9 ion arises from the loss
of 3-amino-2-piperidinone from the initial water loss ion, which
competes with the proline effect to generate the yo6 ion. These
results therefore suggest that the ornithine effect in C-terminal
ornithine containing peptides is stronger than the proline effect
when both are potentially active.
to ornithine makes a profound difference in the fragmentation of
this and similar peptide sequences, as shown in Fig. 4(c). The initial
impact of the presence of ornithine rather than arginine is that
there is less proton sequestration, thereby allowing a more uniform
distribution of cleavages to occur. Nearly full sequence coverage is
obtained with significant abundances of most of the possible b an y
sequence ions. The major water loss apparent in the product ion
spectrum of [GAILDGAILO + H]+ and the prominent b9 that may
arise from the loss of 3-amino-2-piperidinone from the water loss
ion, along with other plausible sequential decomposition products
following water loss, indicate that the ornithine effect is also
prominent in this case.
Peptides containing an internal ornithine
Proteins, non-tryptic peptides and tryptic peptides with missed
cleavages frequently contain arginine residues at intermediate
locations in the amino acid sequence. The data of Fig. 1 indicate
that the ornithine effect is operative when arginine residues at
intermediate locations in the sequence are deguanidinated.
Human Angiotensin II, DRVYIHPF, was examined to explore the
contribution of the ornithine effect at an intermediate location
in the sequence when the aspartic acid and proline effects are
also possible.
As illustrated in Fig. 4(a), the aspartic acid effect can be
preponderant in the absence of a mobile proton, such as is the
case when an ionizing proton is sequestered at an arginine
residue. CID of [DRVYIHPF + H]+ (Fig. 5(a)) results largely in
the formation of the y7 product ion, corresponding to cleavage
C-terminal to aspartic acid. The loss of water from the precursor
may arise from a number of sites, such as, for example, the
carboxylic acids of the C-terminus or aspartic acid side chain or
the hydroxyl group of tyrosine. Conversion of the arginine to
ornithine significantly alters the fragmentation behavior. CID of
[DOVYIHPF + H]+ (Fig. 5(b)) shows selective cleavage C-terminal
to ornithine, as reflected by the highly abundant y6 ion. Other
y ions were also observed at much lower abundance, and a
relatively small b6 ion, which is consistent with the proline effect,
The aspartic acid effect tends to be prominent when there is
relatively little proton mobility in peptides that contain an aspartic
acid residue. In this case, cleavage is favored at the C-terminal
side of aspartic acid. This effect is clearly evident in the CID of
[GAILDGAILR + H]+, shown in Fig. 4(a). The y5 ion, produced by
fragmentation C-terminal to aspartic acid, is essentially the only
fragment ion observed. Figure 4(b) provides a 25Â magnification
of this CID spectrum, revealing that other sequence ions can be
generated, but in much lower abundance. Conversion of arginine
J. Mass Spectrom. 2013, 48, 856–861
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W. M. McGee and S. A. McLuckey
lactam ring is less favored, which presumably underlies the much
weaker effect with lysine residues. The lactam terminated b-ion
can undergo a facile sequential cleavage to generate a smaller
b-ion and loss of 3-amino-2-piperidinone. The ornithine-related
cleavage appears to be even more favored than other selective
cleavages known for CID of peptide cations, such as those N-terminal
to proline and C-terminal to aspartic acid. The presence of an
ornithine residue in a polypeptide cation introduces a particularly
‘weak spot’ for CID.
Selective cleavages of peptide ions can be a complication
when the objective is to generate full peptide sequence.
However, there may be instances in which selective cleavages
can be useful because they introduce a degree of specificity that
is analogous to that associated with selective enzymes in solution.
The common gas phase selective cleavages, however, lack
generality. That is, certain conditions must be met before they
are particularly dominant (e.g. mobile protons for the proline
effect, lack of mobile protons for the aspartic acid effect). For this
reason, a practical approach to top–down protein identification
that takes advantage of selective cleavages has not been developed.
The ornithine effect may prove to be more general. At least, this
appears to be the case based on the peptides we have examined
to date. The conversion of arginine to ornithine via a simple
solution-phase reaction is a relatively simple way to introduce
ornithine into a polypeptide, which both replaces a highly basic
site with a less basic site and opens up the facile cleavage channel
associated with ornithine. We are currently exploring alternate
means for converting arginine residues to ornithine residues
as well as analytical strategies that take advantage of the
ornithine effect.
Figure 5. Comparison of CID of Angiotensin II (human), DRVYIHPF and
ornithinated DOVYIHPF. (a) CID of [DRVYIHPF + H]+; (b) CID of
[DOVYIHPF + H]+; (c) CID of [DRVYIHPF + 2H]+; (d) CID of [DOVYIHPF + 2
H]+. Water loss is labeled with a degree sign (ꢀ). Precursor ion fragmented
(M) is denoted with a lightning bolt ( ).
appeared in the spectrum. The comparison of Figs. 5(a) and 5(b)
shows that deguanidination of angiotensin II diminishes the
aspartic acid effect and opens up the ornithine effect. This is
interpreted to result from the replacement of the highly basic
arginine residue with ornithine, which opens up the dissociation
pathway for formation of the favored six-membered lactam ring.
When angiotensin II is doubly protonated, the proline effect
gives rise to the predominant fragmentation channel, as illustrated
in Fig. 5(c). CID of [DRVYIHPF+ 2H]2+ leads to b6 and y2 as the most
abundant fragments, which arise from cleavage between the
histidine and proline residues. The aspartic acid effect also appears
to contribute as reflected by the appearance of the y27+ ion. An
internal fragment, singly protonated RVYIH, and its water loss
product are also observed and appear to arise from sequential
cleavages arising from both the proline and aspartic acid effects
(i.e. cleavage N-terminal to proline and C-terminal to aspartic acid).
The proline effect becomes important for the doubly protonated
peptide while it is not important for the singly protonated peptide
presumably due to the second proton being sufficiently mobile to
transfer to the tertiary nitrogen of the proline amide nitrogen.
Following conversion of arginine to ornithine, CID of the doubly
protonated peptide, [DOVYIHPF + 2H]2+, results in a dramatic
change in fragmentation behavior, as shown in Fig. 5(d). The
product ion spectrum is dominated by fragments generated from
cleavage C-terminal to ornithine (i.e. the y6, b2 and bo2 ions) with
relatively minor products from cleavages at other amide linkages.
From the standpoint of proton mobility, there is no obvious reason
why the proline effect would be diminished by replacing arginine
with ornithine. It appears that the ornithine effect is clearly favored
over the proline effect when both are potentially active.
Acknowledgement
This work was supported by the National Institutes of Health
under Grant GM 45372.
Supporting information
Supporting information may be found in the online version of
this article.
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Conclusions
The presence of an ornithine residue in a polypeptide cation
introduces a facile peptide backbone cleavage channel C-terminal
to ornithine via the formation of a stable six-membered lactam ring
terminated b-ion. The complementary product is a water molecule
when the ornithine is present at the C-terminus or a y-ion in all
other cases. The mechanism for this ‘ornithine effect’ is also open
to lysine residues. However, the formation of a seven-membered
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J. Mass Spectrom. 2013, 48, 856–861

The ornithine effect: selective cleavages observed in ornithinated peptides
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J. Mass Spectrom. 2013, 48, 856–861
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