Radical Conversion and Migration in Electron Capture Dissociation

Abstract

Electron capture dissociation (ECD) is an important analytical technique which is used frequently in proteomics experiments to reveal information about both primary sequence and post-translational modifications. Although the utility of ECD is unquestioned, the underlying chemistry which leads to the observed fragmentation is still under debate. Backbone dissociation is frequently the exclusive focus when mechanistic questions about ECD are posed, despite the fact that numerous other abundant dissociation channels exist. Herein, the focus is shifted to side chain loss and other dissociation channels which offer clues about the underlying mechanism(s). It is found that the initially formed hydrogen abundant radicals in ECD can convert quickly to hydrogen deficient radicals via a variety of pathways. Dissociation which occurs subsequent to this conversion is mediated by hydrogen deficient radical chemistry, which has been the subject of extensive study in experiments which are independent from ECD. Statistical analysis of fragments observed in ECD is in excellent agreement with predictions made by an understanding of hydrogen deficient radical chemistry. Furthermore, hydrogen deficient radical mediated dissociation likely contributes to observed ECD fragmentation patterns in unexpected ways, such as the selective dissociation observed at disulfide bonds. Many aspects of dissociation observed in ECD are easily reproduced in well-controlled experiments examining hydrogen deficient radicals generated by non-ECD methods. All of these observations indicate that when considering the means by which electron capture leads to dissociation, hydrogen deficient radical chemistry must be given careful consideration.

Full text

rXXXX American Chemical Society Adx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
ARTICLE
pubs.acs.org/JACS
Radical Conversion and Migration in Electron Capture Dissociation
Benjamin N. Moore, Tony Ly, and Ryan R. Julian
Department of Chemistry, University of California, Riverside, California 92521, United States
b
SSupporting Information
’INTRODUCTION
Electron capture dissociation (ECD) and the related method
electron transfer dissociation (ETD) continue to be important
methods for extracting information in mass spectrometry based
proteomics experiments.
15
These techniques are employed to
obtain sequence information from peptides and proteins. Impor-
tantly, highly labile post-translational modifications are typically
preserved during ECD/ETD,
6
which simplifies identification
relative to collisional activation methods.
7
Technological develop-
ments have also enabled ECD/ETD to be used in high-throughput
proteomic analyses, further increasing their utility.
8,9
Despite the
value and popularity of ECD, the underlying mechanisms which
dictate how fragmentation occurs are still not entirely clear and
have been a subject for discussion over the last 10 years.
1,1013
Several of the mechanisms proposed for backbone dissocia-
tion in ECD proceed through an aminoketyl radical structure
which dissociates into c and z
•
ions, as shown in Scheme 1.
Formation of c/z
•
ions in ECD is analytically useful, due to the
consistently high peptide sequence coverage in comparison to
that of b/y ions generated by collision-induced dissociation
(CID). Although the formation of the aminoketyl radical and
subsequent dissociation into backbone fragments has been
studied previously in detail,
14,15
less attention has been given
to other dissociation channels which are commonplace in ECD.
For example, a variety of neutral losses from the charge-reduced
precursor ion are observed in ECD.
1619
These neutral losses
occur frequently and may provide additional insight into the
mechanism of ECD which may not be apparent from an
examination of backbone dissociation alone. Indeed, alternatives
to c/z
•
ion formation exist even for the aminoketyl radical itself,
which can also potentially undergo radical migration, as shown in
Scheme 1b. It has also been postulated that a radical cascade
mechanism may be responsible for some of the fragmentation
observed in ECD.
20
Radical cascade mechanisms require an
initial backbone c/z
•
dissociation and are similar in theory to
dissociation observed from activation of z
•
product ions.
21,22
Neutral loss of amino acid side chains is not exclusive to ECD
but is also prevalent in experiments involving what are referred to
as “hydrogen deficient”radical species. Hydrogen deficient radi-
cals are distinct in that they have one less hydrogen atom than a
Scheme 1. (a) Prototypical Backbone Dissociation into c/z
•
Ions and (b) Migration of the Aminoketyl Radical
Received: October 27, 2010
ABSTRACT: Electron capture dissociation (ECD) is an important
analytical technique which is used frequently in proteomics experi-
ments to reveal information about both primary sequence and post-
translational modifications. Although the utility of ECD is unques-
tioned, the underlying chemistry which leads to the observed fragmentation is still under debate. Backbone dissociation is frequently
the exclusive focus when mechanistic questions about ECD are posed, despite the fact that numerous other abundant dissociation
channels exist. Herein, the focus is shifted to side chain loss and other dissociation channels which offer clues about the underlying
mechanism(s). It is found that the initially formed hydrogen abundant radicals in ECD can convert quickly to hydrogen deficient
radicals via a variety of pathways. Dissociation which occurs subsequent to this conversion is mediated by hydrogen deficient radical
chemistry, which has been the subject of extensive study in experiments which are independent from ECD. Statistical analysis of
fragments observed in ECD is in excellent agreement with predictions made by an understanding of hydrogen deficient radical
chemistry. Furthermore, hydrogen deficient radical mediated dissociation likely contributes to observed ECD fragmentation
patterns in unexpected ways, such as the selective dissociation observed at disulfide bonds. Many aspects of dissociation observed in
ECD are easily reproduced in well-controlled experiments examining hydrogen deficient radicals generated by non-ECD methods.
All of these observations indicate that when considering the means by which electron capture leads to dissociation, hydrogen
deficient radical chemistry must be given careful consideration.

Bdx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
Journal of the American Chemical Society ARTICLE
peptide in a given charge state would typically have: i.e., [(M-H)
•
þnH]
nþ
. These radicals can be produced by various methods
such as gas-phase photodissociation (PD),
23,24
CID of anions,
25
electron detachment dissociation,
26,27
electron-induced dissocia-
tion,
28
CID of nitroso-containing peptides,
29
CID of peroxyca-
rbamates,
30
free radical initiated peptide sequencing (FRIPS),
31
dissociation of peptidemetal adducts,
32,33
and femtosecond
laser pulses.
34
ECD itself also produces c
•
and z
•
type product
ions which are hydrogen deficient radical species and behave as
such.
16,3540
Hydrogen deficient radical peptides generated by
these methods typically yield amino acid side chain losses and a/x
•
backbone fragmentation when subjected to CID or radiative
heating.
41
ECD, on the other hand, typically produces c/z
•
fragments and side chain losses, some of which are distinct to
those observed in hydrogen deficient radical experiments.
A major hallmark of hydrogen deficient radical chemistry is
radical migration, a process which allows the radical to travel to
stable sites within the molecule.
42,43
In radical migration an
initially formed hydrogen deficient radical abstracts a neighbor-
ing hydrogen atom through space, thus quenching one radical
and creating a new one, as shown in Scheme 2a. Radical
migration pathways allow a variety of side chain losses and
backbone dissociations to occur at sites distant from the location
of the initial radical. We have previously proposed that radical
migration pathways in peptides and proteins are determined by
the bond dissociation energies (BDEs) of available hydrogens as
well as the constraints imposed by the gas-phase structure.
41
Migration is facilitated when the BDE of the radical acceptor site
is comparable to or lower than the BDE of the radical donor.
Conformational constraints must also allow proper alignment of
the donor and acceptor in order for migration to occur. Under
conditions where the relative BDE difference is favorable and
structural constraints are negligible, barriers to migration are
predicted to be quite small.
43,44
Typically we have found that the
flexibility available in moderately sized peptides minimizes
structural constraints such that the BDEs become good qualita-
tive predictors of radical mobility.
41
This is not the case in whole
proteins, where radical fragmentation is confined to spatially
adjacent residues, although valuable structural information can
be obtained in this fashion.
45
At face value, backbone fragmentation and side chain losses
from the charge-reduced precursor ion in ECD appear to be
distinct from what is observed in hydrogen deficient radical
experiments, because many of the products have been mass-
shifted. Formally, fragmentation in ECD is initiated from a
“hydrogen abundant”radical species, i.e. [(M þH
•
)þnH]
nþ
,
which after charge reduction has one extra hydrogen atom
relative to the canonical protonated species [M þnH]
nþ
. The
hydrogen abundant product that is formed after electron capture
becomes the initial structure leading to all observed ECD
fragmentation. Electron capture, as proposed by McLafferty,
Zubarev, and co-workers
1
in what is referred to as the Cornell
mechanism, occurs directly at a protonated charge site, resulting
in a hydrogen abundant radical species. This species can then
donate a hydrogen atom to a backbone carbonyl to form the
aminoketyl radical and subsequently undergo c/z
•
type fragmen-
tation. Charge remote electron capture, as proposed by
Turecek
46
and Simons,
11,47
occurs by electron capture at a
backbone carbonyl or disulfide to form an anionic species.
Dissociation can then occur at the site of electron capture directly
(Utah mechanism) or after proton transfer (Washington me-
chanism). Numerous charge remote capture pathways involving
backbone or disulfide bond cleavage are frequently referred to
collectively as the UtahWashington mechanism. The Cornell
and Washington mechanisms both involve conversion to a
hydrogen deficient radical species; however, the possibility of
radical migration within this species is not typically considered.
For simplicity, we will adopt the formalism of direct electron
capture at charged sites to describe chemistry related to hydrogen
deficient radical migration in the present paper. It should be
understood that, in theory, all of the chemistry that will be described
could also be initiated by charge remote electron capture followed by
proton transfer. Determining the extent to which this might occur
in actual ECD is beyond the scope of this paper.
It is important to point out that hydrogen abundant radical
species can also undergo radical migration, but not by the
hydrogen atom abstraction process previously described for
hydrogen deficient radicals. Instead, the excess hydrogen atom
is transferred from the initial radical site to a hydrogen atom
accepting site to form a new hydrogen abundant radical at that
location, as shown in Scheme 2b. In this case, the radical and
hydrogen atom are collocated and move in the same direction,
which is in contrast with hydrogen deficient migration, where the
hydrogen atom and radical migrate in opposite directions. This
subtle difference in the mechanism of migration is an important
characteristic in distinguishing hydrogen abundant and hydrogen
deficient radical chemistry.
Hydrogen abundant radical migration should also not be
confused with “radical conversion”, where a hydrogen abundant
radical is converted into a hydrogen deficient radical. An example
is shown in Scheme 2c, where the aminoketyl radical, a hydrogen
deficient product, is formed from the conversion of a hydrogen
abundant precursor. Transfer of the abundant hydrogen from the
charge-reduced hypervalent amine transforms the backbone
carbonyl into an alcohol, thus converting the radical from
hydrogen abundant to hydrogen deficient. A loss of a degree of
saturation accompanies this process.
In this paper we demonstrate that hydrogen deficient radical
chemistry plays an important role in much of the dissociation that
is observed in ECD. Statistical analysis of a large body of ECD
data suggests that the hydrogen-abundant species that are
initially formed by electron capture quickly convert into hydro-
gen deficient radicals. This can occur via a variety of pathways;
Scheme 2. Generic Mechanisms of (a) Hydrogen Deficient
Radical Migration, Where the Radical and Hydrogen Atom
Exchange Locations, (b) Hydrogen Abundant Radical Mi-
gration, Where the Radical and Hydrogen Atom Are Collo-
cated, and (c) Radical Conversion from a Hydrogen
Abundant to a Hydrogen Deficient State

Cdx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
Journal of the American Chemical Society ARTICLE
the most common pathways are outlined herein. Photodissocia-
tion experiments designed to explore side chain dissociations
observed in hydrogen-deficient radical peptides are reported.
These experiments confirm that hydrogen deficient chemistry
can rationalize similar fragmentations observed in ECD, includ-
ing selective fragmentation observed at disulfide bonds. Under-
standing the role that radical chemistry plays in ECD provides
significant insight into the mechanisms which are responsible for
both backbone and side chain dissociation.
’EXPERIMENTAL SECTION
The peptide RPPGYSPFR was purchased from American Peptide Co.
(Sunnyvale, CA). This peptide was iodinated at tyrosine by a previously
described method.
48
The carboniodine bonds of iodotyrosine-con-
taining peptides were photodissociated in the gas phase by transmitting
fourth harmonic (266 nm) laser light, generated from a flash-pumped
Nd:YAG laser (Continuum, Santa Clara, CA), through a quartz window
on the rear vacuum flange of a modified LTQ linear ion trap (Thermo-
Fisher, Waltham, MA) as previously described.
23
R-Methylcysteine was
purchased from Nagase Co. (Osaka City, Japan), Fmoc protected,
49
and
acetamide protected. The peptides YVDIAIPCGNK and its R-methyl-
cysteine derivative were synthesized by solid-phase peptide synthesis.
49
S-Cysteine-acetamide-containing peptides were derived from cysteine-
containing peptides by reaction with an excess of iodoacetamide in water
in the dark at 40 °C for 1 h.
50
Dehydroalanine-containing peptides were
derived from phosphoserine-containing peptides by reaction with Ba-
(OH)
2
solution at 40 °C for 1 h.
51
Disulfide-bonded dipeptides were
created by oxidizing two peptides, each containing a single free cysteine
in the presence of DMSO, 20% by volume in water.
52
Each of these
products was purified separately on a C8 peptide purification column
and diluted to a concentration of 5 μM in 90/10/1 acetonitrile/water/
acetic acid before being electrosprayed.
Chemical structure and energy calculations were performed with
the Gaussian 03 Ver. 6.1 Rev. D.01 software package (Gaussian, Inc.,
Wallingford, CT).
53
All calculations used the hybrid density functional
theory B3LYP with the 6-31G(d) basis set unless otherwise stated. The
bond dissociation energies (BDE) of the neutral aminoketyl radical and
Gln, Glu, Asp, and Asn side chain radicals were calculatedby the isodesmic
reaction method using the experimentally determined BDE of
HCH
2
OH (96.1 (0.2 kcal mol
1
)asareference.
54
All other BDEs
were calculated using appropriate reference energies. Transition states
were determined by quasi-Newtonian synchronous transit (QST3) calcu-
lations and verified by visualization of the single imaginary frequency.
55
Percentages of side chain loss in Table 1 were calculated on the basis
of analysis of the SwedECD database and are similar to those which have
been previously reported.
17
An algorithm was used which examined each
peptide ECD spectrum in the database and looked for specific side chain
losses by mass from the charge-reduced species. The number of peptides
which contained the experimentally observed side chain loss and also
contained the residue expected to generate the side chain loss were
summed and then divided by the total number of peptides containing the
residue. Finally, these values were converted to a percentage and
compiled for all side chain losses in Table 1. Peptides containing amino
acids with side chain losses that overlap by mass were excluded from the
analysis when appropriate.
’RESULTS AND DISCUSSION
Evidence for Hydrogen Deficient Chemistry. It is first nec-
essary to determine whether any of the numerous dissociation
pathways observed in ECD are best explained by hydrogen
deficient radical chemistry. For a statistically relevant source of
ECD data, we will use the SwedECD database made publicly
available by the Zubarev group.
17
SwedECD contains 11 491
ECD mass spectra of peptides derived from a tryptic digest of cell
lysates. From this database we can extract trends relating forma-
tion of certain product ions with amino acid identity, sequence
length, and a variety of other parameters. Specific side chain
losses from the charge-reduced species in ECD have been
previously investigated for their analytical utility in the differ-
entiation of isomeric sequences, improvement of database
searching, and de novo sequencing.
17
In this work, however,
side chain loss will be used to investigate the mechanistic details
of ECD and its relation to hydrogen deficient radical chemistry.
Only side chain loss from the charge-reduced parent ion in ECD
is considered, since this is the product ion that results directly
from electron capture and may initially contain a hydrogen
abundant radical. Side chain losses from other products, such
as z
•
ions, have been examined previously and proceed through
hydrogen deficient radical chemistry.
35
Table 1 shows side chain
losses that are typically observed in both hydrogen abundant and
hydrogen deficient radical experiments. At first glance, most of
the hydrogen deficient/abundant side chain losses in Table 1
appear to be different from each other and unrelated. However,
closer inspection and consideration is warranted, as will be
detailed in the following sections.
Leucine Radical Side Chain Loss. The side chain loss of 56
Da from leucine is observed in both ECD experiments and when
hydrogen deficient radicals are fragmented. However, simple
observation of the same mass loss does not necessarily imply that
the loss occurs via the same mechanism in both experiments;
therefore, likely hydrogen abundant and hydrogen deficient
pathways for the loss of 56 Da are considered in Scheme 3.
The loss of 56 Da represents the mass of nearly the entire side
chain; however, as shown in Scheme 3a, homolytic cleavage of
the appropriate bond would actually result in a loss of 57 Da.
Therefore, any correct mechanism will require the transfer of a
hydrogen atom from the side chain to the peptide prior to
dissociation. Hydrogen abundant dissociation typically involves
attack by the excess hydrogen atom (see Scheme 2b), which is
unlikely in the case of the leucine side chain due to the absence of
favorable sites to receive the hydrogen atom. Furthermore, attack
at the side chain would ultimately result in a loss of 58 Da, as
shown in Scheme 3b, not the required 56 Da. Similarly, hydrogen
atom attack at the Rposition would result in a loss of 57 Da,
which is not acceptable either. Hydrogen abundant radical
chemistry does not therefore offer a palatable explanation for
the observed loss. In contrast, hydrogen deficient chemistry
readily leads to this type of loss via the mechanism outlined in
Scheme 3c.
36
If the initial hydrogen abundant parent ion under-
went conversion to a hydrogen deficient species, then migration
of that radical to the γposition of leucine would provide the
required hydrogen transfer. Subsequent βdissociation would
generate the observed loss of 56 Da. Consideration of all possible
mechanisms suggests that this loss is actually generated by the
same pathway in both ECD and hydrogen deficient radical
experiments. Furthermore, it implies that significant conversion
to a hydrogen deficient state capable of radical migration occurs
within the charge-reduced parent ion in ECD.
S-Cysteine-Acetamide Side Chain Loss and Disulfide Bond
Cleavage. Another side chain loss observed in both ECD and
hydrogen deficient radical experiments is the loss of the S-
cysteine-acetamide side chain. In the SwedECD study, peptides
were reacted with iodoacetamide to cap cysteine residues, thus
forming S-cysteine-acetamide and preventing any possible

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Journal of the American Chemical Society ARTICLE
disulfide bond formation (this is commonly done in proteomics
experiments). As seen in Table 1, peptides containing modified
cysteine have an ∼42% probability of producing a loss of 90 Da in
ECD experiments. This loss is therefore highly favored in ECD.
Hydrogen deficient radical peptides containing modified cy-
steine have not been investigated previously. In order to provide
context for comparison, several peptides were treated with
iodoacetamide to cap the cysteine, iodinated at tyrosine, and
converted into hydrogen deficient radicals by photoactivation.
As shown in Figure 1a, collisional activation of [(CDPGYIGSR)
•
þH]
þ
yields a dominant loss of 90 Da. As discussed previously,
radical migration is influenced by both relative BDEs and peptide
structure. The results in Figure 1a likely result from structural
factors which favor migration of the radical to cysteine; never-
theless, it is clear that the loss of 90 Da can be highly favored
under the proper conditions. It is anticipated that doubly
protonated CDPGYIGSR will not have the same structure as
the singly protonated ion, which should influence radical migra-
tion. Indeed, Figure 1b shows CID of the doubly charged peptide
radical yields a loss of 90 Da, though several other dissociation
channels are observed as well. The results in Figure 1 are similar
to those obtained for several other peptides and confirm that the
Table 1. Partial List of Observed Neutral Side Chain Losses from the Charge-Reduced Peptides in ECD and Observed Neutral
Side Chain Losses from Hydrogen-Deficient Radical Peptides
ECD neutral side chain losses hydrogen deficient radical side chain losses
amino acid chem formula exact mass % peptides with loss
c
amino acid chem formula exact mass
Arg C
4
H
11
N
3
101.0953 2.2 Arg C
4
H
9
N
3
99.0796
Arg CH
3
N2 43.0296 14.3 Arg C
3
H
8
N
3
86.0718
Asn C
2
H
5
NO 59.0371 47.1
a
Asn CH
2
NO 44.0136
Asn CH
2
NO þNH
3
61.0401 13.8 Asp CHO
2
44.9977
Asp C
2
H
4
O
2
60.0211 60.4
a
Cys* C
2
H
4
NO 58.0293
Asp CHO
2
þNH
3
62.0242 2.7 Cys* C
2
H
4
NOS 90.0014
Cys
b
C
2
H
4
NO 58.0293 88.8 Gln C
2
H
4
NO 58.0293
Cys
b
C
2
H
4
NO þNH
3
75.0558 19.9 Gln C
3
H
5
NO 71.0371
Cys
b
C
2
H
4
NOS þNH
3
107.0279 56.3 Glu C
2
H
3
O
2
59.0133
Cys
b
C
2
H
5
NO 59.0371 43.2
a
Glu C
3
H
4
O
2
72.0211
Cys
b
C
2
H
4
NOS 90.0014 42.3 His C
4
H
4
N
2
80.0374
Gln C
2
H
4
NO þNH
3
75.0558 14.8 His C
3
H
3
N
2
67.0296
Gln C
2
H
5
NO 59.0371 5.6
a
Ile C
2
H
5
29.0391
Gln C
3
H
5
NO 71.0371 3.9 Ile C
4
H
8
56.0626
Gln C
3
H
5
NO þNH
3
88.1000 0.3 Leu C
4
H
8
56.0626
Glu C
2
H
3
O
2
þNH
3
76.0393 21.3 Leu C
3
H
7
43.0584
Glu C
3
H
4
O
2
þNH
3
89.0471 6.7 Lys C
3
H
8
N 58.0657
Glu C
2
H
4
O
2
60.0211 1.0
a
Lys C
4
H
9
N 71.0735
His C
4
H
6
N
2
82.0531 38.4 Met C
3
H
6
S 74.0190
Ile C
2
H
5
þNH
3
46.0651 7.2 Met CH
3
S 46.9955
Ile C
4
H
8
þNH
3
73.0891 2.6
a
Met C
2
H
5
S 61.0112
Ile C
4
H
8
56.0626 1.0
a
Ser CH
2
O 30.0106
Ile C
2
H
5
29.0391 0.7 Thr C
2
H
4
O 44.0262
Leu C
4
H
8
þNH
3
73.0891 11.9
a
Trp C
9
H
7
N 129.0578
Leu C
3
H
7
þNH
3
60.0849 11.6 Tyr C
7
H
6
O 106.0419
Leu C
4
H
8
56.0626 4.7
a
Leu C
3
H
7
43.0584 1.4
Met C
3
H
6
SþNH
3
91.0456 17.7
Tyr C
7
H
8
O 108.0575 3.5
a
Peptides containing other residues that exhibit the exact same side chain loss by mass are excluded from these results.
b
Denotes S-cysteine-acetamide.
c
% peptides with loss only considers peptides which contain the residue and is calculated by finding the percentage of peptides which upon ECD give the
corresponding side chain loss within (0.01 Da.
Scheme 3. Leucine Side Chain Loss: (a) Homolytic Cleavage
of the C
R
C
β
Bond Results in 57 Da Loss; (b) Attack by
Hydrogen Atom Results in Loss of 58 Da; (c) Abstraction of
the γ-Hydrogen Yields the Observed Loss of 56 Da

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Journal of the American Chemical Society ARTICLE
loss of 90 Da from modified cysteine is a favorable dissociation
channel in hydrogen deficient radical peptides.
Possible mechanisms for the loss of 90 Da from modified
cysteine are shown in Scheme 4. Homolytic cleavage of the CS
bond in S-cysteine-acetamide would indeed result in the ob-
served 90 Da loss. There are two pathways which can lead to this
loss. The first involves hydrogen atom attack at the β-carbon, as
shown in Scheme 4b. The β-carbon position in amino acids is not
predicted to have a high hydrogen atom affinity, because a
pentavalent carbon intermediate is created, which mitigates
the likelihood of this mechanism. Alternatively, a hydrogen
deficient radical starting from the R-position of the amino acid
is able to rearrange and produce the 90 Da side chain loss as
shown in Scheme 4c. The R-position is known to stabilize
hydrogen deficient radicals due to the captodative effect.
51,54,56,57
The results in Figure 1 clearly demonstrate that the 90 Da
loss mechanism in Scheme 4c is favorable; however, further
experiments are needed to prove that the same pathway is
operative in ECD. In order to probe this issue experimentally,
two peptides with the sequence YVDIAIPcGNK were prepared,
one of them containing an R-methylcysteine variant. The R-
methyl group should block the pathway outlined in Scheme 4c
(because the abstracted hydrogen is replaced with a methyl
group) while leaving the pathway in Scheme 4b open. The results
in Figure 2a illustrate that the expected loss of 90 Da is observed
in the standard peptide. In Figure 2b however, the R-methyl
variant does not exhibit a loss of 90 Da, confirming that
this fragmentation proceeds via the mechanism outlined in
Scheme 4c in ECD experiments.
If hydrogen deficient radicals access the R-positions of amino
acids following electron capture, radical conversion, and migra-
tion, then why is dissociation at modified cysteine observed while
other comparable side chain losses are not? The most plausible
hypothesis relates to the stability of the products, which is known
to influence transition state energies in hydrogen deficient radical
dissociation. The BDEs for all possible side chain losses that can
occur from the R-radical initiation are shown in Figure 2c. The
range of BDEs for all R-radicals is highlighted in red.
54
Only the
loss of modified cysteine yields a radical which is comparable in
stability to the R-radicals. All other side chain losses are therefore
predicted to be significantly endothermic and disfavored. When
taken as a whole, it is clear that consideration of the relevant
energetics from a hydrogen deficient radical chemistry perspec-
tive explains both the favorable loss of 90 Da from modified
cysteine and the absence of other equivalent side chain losses
from the remaining amino acids.
Scheme 4. S-Cysteine-Acetamide Side Chain Loss: (a)
Homolytic Cleavage of the C
β
S Bond Results in 90 Da Loss;
(b) Hydrogen Atom Transfer to the β-Carbon Could Produce
the 90 Da Loss; (c) Abstraction of the R-Hydrogen Yields the
Experimentally Observed 90 Da Loss
Figure 2. (a) ECD spectrum of the peptide YVDIAIPcGNK. (b) ECD
spectrum of the peptide YVDIAIPc*GNK, where c* denotes R-methyl-
cysteine. (c) BDEs of the products resulting from R-radical driven side
chain dissociation from standard amino acids and the nonstandard
amino acid S-cysteine-acetamide. The red region is the BDE range of
amino acid R-hydrogens.
Figure 1. (a) PD/CID of singly charged [(cDPGYIGSR)
•
þH]
þ
and
(b) PD/CID of doubly charged [(cDPGYIGSR)
•
þ2H]
2þ
. The lower
case “c”denotes the amino acid S-cysteine-acetamide.

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Journal of the American Chemical Society ARTICLE
Disulfide Bonds. The specific cleavage of disulfide bonds by
ECD is a well-known process which is thought to occur via one of
two pathways: direct electron capture into an antibonding orbital
of the disulfide bond or hydrogen atom attack at one of the sulfur
atoms.
3,11
Both of these mechanisms may be occurring in ECD,
but a hydrogen deficient radical pathway also exists and may
contribute to the observed disulfide cleavage products. The
singly disulfide bound complex resulting from combination of
the peptides RPHERNGFTVLCPKN and VCYDKSFPISHVR
(iodinated at tyrosine) was subjected to photodissociation to
generate a hydrogen deficient radical system. Collisional activa-
tion of the radical product is shown in Figure 3a. Cleavage of the
disulfide bond leads to the major products. Again, the highly
dominant disulfide dissociation is likely due to favorable struc-
tural features which place the disulfide and nascent radical in
close proximity. When the even-electron peptide complex is
subjected to CID, no disulfide dissociation is observed. For
comparison, a similar experiment with the peptides SLRRS-
SCFGGR and CDPGYIGSR (iodinated at tyrosine) is shown
in Figure 3b. In this case, dissociation at the disulfide is less
dominant and is accompanied by related losses stemming from
cleavage of the CS bond adjacent to the disulfide. The
hydrogen deficient pathways which lead to these products are
shown in Scheme 5. Cleavage of the disulfide bond can be
initiated from attack at the β-position or directly at the disul-
fide.
58
In contrast, dissociation of the CS bond is initiated from
the more stable R-position. The data in Figure 3 clearly confirms
that both pathways are possible in hydrogen deficient radical
peptides. Both types of dissociation are also observed in actual
ECD experiments.
3
Given that we have established that hydro-
gen deficient radicals are generated in ECD experiments, it is
likely that some portion of the dissociation observed at disulfide
bonds occurs via the pathways outlined in Scheme 5. The
observed preference for dissociation at disulfide bonds in ECD
may be due to the existence of multiple feasible fragmentation
pathways.
Origin of Hydrogen Deficient Radical Chemistry in ECD.
The dissociation products observed in ECD from leucine, S-
cysteine-acetamide, and disulfide bonds are consistent with
pathways that would be expected from hydrogen deficient radical
chemistry. Some of the observed fragmentations do not have
plausible hydrogen abundant radical pathways, such as the loss of
56 Da from leucine. It is therefore reasonable to conclude that a
significant population of the radicals in charge-reduced parent
ions of ECD are converted to hydrogen deficient radials. We will
now discuss the mechanisms by which hydrogen deficient
radicals are created in ECD.
Direct Electron Capture at Charged Sites. NH
3
loss can occur
promptly after direct electron capture and is a commonly
observed dissociation in ECD experiments. Loss of NH
3
con-
comitantly converts the peptide to a hydrogen deficient state,
leaving behind a radical at the point of dissociation.
17
It is not
surprising, therefore, to note that all of the NH
3
accompanied
side chain losses listed in Table 1 are readily explained in terms of
hydrogen deficient chemistry. Importantly, NH
3
-accompanied
side chain losses in ECD experiments represent a substantial
fraction of the total listed in Table 1; therefore, many of the side
chain losses observed in ECD clearly occur via hydrogen
deficient chemistry, but this fact is cloaked by the loss of NH
3
,
which shifts the masses from the equivalent losses on the right
side of Table 1.
If direct electron capture occurs on a doubly charged tryptic
peptide, the most likely sites for capture are arginine, lysine, and
the N-terminus. Direct electron capture at protonated lysine is
shown in Scheme 6a. A hydrogen abundant radical species is
created in the form of a hypervalent NH
3
group. A similar
result will be produced by direct electron capture at the N-ter-
minus. In contrast, direct electron capture at a protonated
arginine residue, as shown in Scheme 6b, yields a hydrogen
deficient radical species directly via a loss of a degree of saturation
in the guanidinium moiety. This conversion has been noted
previously in quantum mechanical structure calculations.
59
The
capacity of arginine to convert directly to a hydrogen deficient
radical should lead to observable differences in the dissociation
patterns for arginine-containing peptides versus lysine-contain-
ing peptides if there is a competition between hydrogen abun-
dant and hydrogen deficient radical chemistry as postulated
herein. The extent to which this difference is observable will also
be influenced by the process of H atom loss.
Following direct electron capture, conversion to an even-
electron species is possible through the loss of an H atom for
arginine, lysine, and the N-terminus. The even-electron product
left behind by H atom loss no longer contains a radical and thus
cannot undergo subsequent radical chemistry. Hydrogen atom
loss from the charge-reduced ion is commonly observed in ECD.
Figure 3. (a) PD/CID of the disulfide-bound peptides (A) VCYDKSF-
PISHVR and (B) RPHERNGFTVLCPKN. (b) PD/CID of the disul-
fide-bound peptides (A) SLRRSSCFGGR and (B) CDPGYIGSR.
Scheme 5. Hydrogen Deficient Radical Disulfide Fragmen-
tation: (a) Disulfide Bond Cleavage; (b) CS Bond
Dissociation

Gdx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
Journal of the American Chemical Society ARTICLE
One factor that strongly influences H atom loss is peptide size,
1
which is demonstrated in Figure 4a. The size effect can be
rationalized in a generic fashion by consideration of peptide
structure. For doubly charged peptides, shorter sequences will
exhibit less intramolecular charge solvation due to increased
Coulombic repulsion relative to longer peptides. The results in
Figure 4a clearly indicate that as charge solvation becomes more
favorable in larger peptides, H atom loss is observed less
frequently. This further suggests that persistence of the products
formed in Scheme 6 likely requires significant charge solvation,
perhaps to remove energy released from the electron capture
event or to facilitate hydrogen atom transfer.
The pathway shown in Scheme 6b implies that arginine may be
less susceptible to H atom loss than lysine because arginine can
directly access a hydrogen deficient state. Figure 4b shows the
individual and averaged values of the ratio of charge reduced to H
atom loss intensity as a function of peptide size for arginine- and
lysine-containing peptides from the ECD database. Although the
predominance of z ions in the database suggests that charge
capture at the N-terminus may occur more commonly than at
lysine or arginine, a clear trend is still observed. The results
demonstrate that arginine-containing peptides are less likely to
undergo H atom loss compared to lysine, particularly for larger
peptides. As a control, a similar analysis was performed for
alanine and glycine, which are residues not associated with direct
electron capture. No difference is observed between alanine and
glycine, as shown in Figure 4c (the statistical sampling breaks
down for the largest peptides due to low numbers). These results
suggest that the pathway in Scheme 6b is one viable route by
which electron capture can lead to a hydrogen deficient state.
Radical Conversion at the Peptide Backbone. The product
generated by direct electron capture at lysine shown in
Scheme 6a can be converted into a hydrogen deficient radical
by several routes. Scheme 2c depicts hydrogen atom transfer
from a hydrogen abundant moiety to a backbone carbonyl on a
peptide. This isobaric conversion from a hydrogen abundant to a
hydrogen deficient radical involves loss of a degree of saturation
and forms an aminoketyl radical structure. The aminoketyl
radical has been extensively studied by many groups as the
proposed immediate precursor to c/z
•
ion formation; however,
the radical structure is hydrogen deficient and is also theoretically
capable of hydrogen abstraction. The calculated BDE of the
aminoketyl radical structure, 365 kJ/mol, is higher than the
average R-position BDE of ∼350 kJ/mol, which suggests that
radical migration from the aminoketyl to an R-position would be
a reasonable process. Direct synthesis of this radical structure in
the gas phase would therefore be extremely useful in answering
questions about the propensity of radical migration versus c/z
•
formation from the aminoketyl radical. Hydrogen deficient
radical peptides naturally dissociate via pathways which generate
aminoketyl-like radical structures, as shown in Scheme 7.
Figure 5a shows the CID of an aminoketyl radical generated by
the C-terminal pathway shown in Scheme 7c for the peptide
RPPGYSPFR. A significant amount of c
8
ion is formed at the
C-terminus, in agreement with the anticipated dissociation site
for the aminoketyl radical shown in Scheme 7c. Hydrogen
deficient radical initiated side chain losses such as 86 and 99
Da from arginine are also observed in significant abundance. The
fragment ions a
5
and a
8
are also products typical of hydrogen
deficient backbone dissociation due to radical migration to the β-
positions of aromatic residues in the peptide.
41
Finally, the c
5
fragment is related to an aminoketyl mechanism through serine,
which does not have an isolatable intermediate.
41
Similar results
are obtained with aminoketyl radicals generated in peptides by
the mechanisms shown in Scheme 7a,b, although the relative
Figure 4. (a) Abundance of hydrogen atom side chain loss as a function
of peptide length. The hydrogen atom loss is shown as the fractional
abundance relative to the precursor ion. Each peptide is represented as a
data point. Histograms for (b) arginine- and lysine-containing peptides
and (c) alanine- and glycine-containing peptides. Ratios of charge-
reduced parent ion abundance versus hydrogen atom loss after electron
capture are plotted as a function of peptide length. In all plots the average
values for each peptide length are shown as large squares.
Scheme 6. (a) Protonated Lysine and (b) Protonated
Arginine Side Chains after Direct Electron Capture at the
Charge Site

Hdx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
Journal of the American Chemical Society ARTICLE
amount of c/z
•
type dissociation that is observed is reduced.
Importantly, c/z
•
ions are observed adjacent to the expected
location of the aminoketyl radical in every case. The relative
yields of c/z
•
ion formation versus hydrogen deficient radical
migration observed in Figure 5a and similar experiments are not
in agreement with results obtained in ECD, where the formation
of c/z
•
ions typically dominates.
As stated above, the products in Scheme 7 are similar to, but
not identical with, the aminoketyl radicals generated in ECD.
The primary difference relates to the presence of dehydroalanine.
As noted previously,
60
dehydroalanine allows delocalization of
the radical produced by the pathway in Scheme 7b and interferes
with c/z
•
ion product formation for the aminoketyl radicals
generated by the pathways in Scheme 7a,c. Therefore, it is not
surprising that modulation of the aminoketyl radical chemistry
enables additional hydrogen deficient radical migration to occur.
In order to confirm the relevance of the results in Figure 5a, we
conducted further experiments to determine if the presence of
dehydroalanine would influence actual ECD experiments. In
Figure 5b the side chain loss region of the ECD spectrum for a
phosphoserine-containing peptide is shown. Typical c/z
•
frag-
mentation is observed elsewhere in the spectrum, and most of the
side chain losses correspond to hydrogen abundant pathways, as
can be seen in Table 1. Figure 5c shows the ECD spectrum for
the same peptide where the phosphoserine has been chemically
converted to dehydroalanine. Hydrogen deficient side chain
losses from the parent ion such as 99 and 86 Da from arginine
along with 56 and 43 Da from leucine are observed and match by
exact mass with those given for hydrogen deficient processes in
Table 1. A significant decrease in c/z
•
ion formation in the
dehydroalanine-containing peptide was also observed. These
results suggest that modulation of the aminoketyl radical can
influence the propensity of this intermediate to undergo c/z
•
ion
formation versus radical migration. It is worth noting that
structural effects due to the absence of phosphate may also
contribute to the observed differences in dissociation in Figure 5.
It is likely that peptide sequence, location of charges, hydrogen-
bonding networks, and other factors influence the microenviron-
ment for each potential aminoketyl radical in a peptide. Similarly,
it is likely that some fraction of these aminoketyl radicals will
undergo migration and are therefore a potential source of
hydrogen deficient chemistry observed in ECD.
Radical Conversion at Amino Acid Side Chains. Hydrogen
atom transfer to the amide side chains of glutamine and
asparagine (as noted previously),
61
and S-cysteine-acetamide
produces radical structures similar to the aminoketyl backbone
radical shown in Scheme 1. Analysis of the fragmentation of these
side chains is therefore directly relevant to the backbone dis-
sociation typically observed in ECD. As shown in Table 1,
analysis of the SwedECD database reveals that 47.1% of Asn-
containing peptides yield a side chain loss of 59 Da following
hydrogen atom transfer, as shown in Scheme 8a. This pathway is
highly analogous to backbone dissociation, which produces c/z
•
ions. Although a similar mechanism can be drawn for glutamine
as shown in Scheme 8b, only 5.6% of peptides containing
glutamine actually exhibit this loss. The disparity can be ratio-
nalized by the stabilities of the nascent radicals. For asparagine, a
highly stabilized R-radical is the product, whereas for glutamine a
much less stable primary carbon radical is generated. The
calculated transition state energies for the pathways in
Scheme 8a,b are also in excellent agreement with the experi-
mental observations. The transition state energy for asparagine is
∼46 kJ/mol, and the loss is somewhat endothermic (ΔH=37
kJ/mol) due to disruption of hydrogen bonding with the side
chain. In contrast, the transition state energy for glutamine is
∼103 kJ/mol and the reaction is endothermic by 137 kJ/mol.
Figure 5. (a) CID of the aminoketyl radical product from PD/CID of
the peptide RPPGY
I
SPFR. (b) ECD side chain loss data for RLEA-
sLADVR; hydrogen abundant initiated losses are shown in italics. (c)
ECD side chain loss data for RLEAdLADVR; hydrogen deficient losses
are shown in boldface. The lower case “s”represents phosphorylated
serine, and “d”represents dehydroalanine.
Scheme 7. The Three Pathways Which Can Generate Ami-
noketyl Radicals by Radical Migration: (a) Aspartic Acid
Pathway I; (b) Aspartic Acid Pathway II; (c) C-Terminal
Pathway

Idx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
Journal of the American Chemical Society ARTICLE
This rationale is further supported by the observation that the
same loss of 59 Da from S-cysteine-acetamide occurs with 43.2%
regularity and produces a stable sulfur radical
62
as the product.
These observations are in excellent agreement with the trends
observed for backbone dissociation with the introduction of
dehydroalanine into peptides as shown in Figure 5: i.e., dissocia-
tion from an aminoketyl radical can be inhibited by destabilizing
the radical product.
A closely related side chain loss of 60 Da can occur for aspartic
and glutamic acid, as shown in Scheme 9. The relative frequen-
cies are 60.4% and 1.0%, respectively. Again, the results can be
rationalized in terms of the stabilities of the radical products.
63
Although side chain dissociation is disfavored in the cases of Glu
and Gln, a stable radical species is formed on the carbonyl carbon,
which may then undergo hydrogen deficient radical migration.
The initially formed side chain radicals have calculated BDEs of
371 kJ/mol for Glu and 356 kJ/mol for Gln, which are both
expected to be able to migrate to lower energy R-radical
positions. These radicals are likely formed on Glu and Gln in
high abundance (on the basis of the amount of dissociation
observed for Asp and Asn) and are likely sources of subsequent
hydrogen deficient radical chemistry in peptides containing these
residues.
Charge remote electron capture at acidic sites (such as the side
chains of glutamic and aspartic acid) has not been evaluated
previously and may differ substantially from capture at amide
sites. It is therefore unknown presently whether charge remote
capture analogous to what is predicted at backbone amide
sites would be feasible for the side chains of Glu and Asp.
Regardless of whether such charge remote capture is subse-
quently shown to be unlikely, the results can still be rationalized
on the basis that Glu and Asp side chains are excellent hydrogen
bond partners and are therefore likely to be solvating charges in
peptides. This would make them likely targets for H atom attack
following electron capture at a protonated site. Indeed, hints of
such behavior can be observed in statistical analysis of ECD
fragmentation.
64
’CONCLUSION
Many pathways exist for the conversion of hydrogen
abundant radicals to hydrogen deficient radicals in ECD. This
conversion is the key to understanding a number of side chain
losses which are not readily explained by hydrogen abundant
processes alone. Side chain losses such as 56 Da from leucine
and 90 Da from S-cysteine-acetamide in ECD point to both the
creation and migration of hydrogen deficient radicals in the
charge reduced parent ion. Examination of the SwedECD
database also shows that hydrogen deficient side chain losses
are regularly observed after a radical conversion process has taken
place, such as the neutral loss of NH
3
. The propensity of
hydrogen atom loss from the charge-reduced parent ion in
ECD is directly related to the ability of a hydrogen abundant
species to convert to a hydrogen deficient species. As expected,
the amount of hydrogen atom loss is therefore dependent
on the structure of the charge site as well as the length of the
peptide in which it resides. From this information it can be
concluded that electron capture can lead directly to a hydrogen
deficient species or an initially formed hydrogen abundant radical
may quickly convert to a hydrogen deficient radical, which then
dictates much of the subsequent observed chemistry that leads to
dissociation.
’ASSOCIATED CONTENT
b
SSupporting Information. Figures giving Asn and Glu
transition state structures and text giving the full citation for ref
52. This material is available free of charge via the Internet at
http://pubs.acs.org.
’AUTHOR INFORMATION
Corresponding Author
ryan.julian@ucr.edu
’ACKNOWLEDGMENT
We thank Joe Loo for instrument time at UCLA to acquire the
ECD data. We thank Jack Simons, Jack Beauchamp, Evan
Williams, Julia Laskin, and Frank Turecek for helpful discussions.
This work was supported by funds from the National Science
Foundation (No. CHE-0747481).
Scheme 9. Hydrogen Atom Attack at (a) Asp and (b) Glu
Scheme 8. Hydrogen Atom Attack at (a) Asn, (b) Gln, and
(c) S-Cys-Acetamide

Jdx.doi.org/10.1021/ja1096804 |J. Am. Chem. Soc. XXXX, XXX, 000–000
Journal of the American Chemical Society ARTICLE
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