Tandem MS analysis of selenamide-derivatized peptide ions

Article abstract of DOI:10.1007/s13361-011-0170-4

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se-S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se-S cleavage, analogous to the S-S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se-S of the tag to the S-S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se-S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

Full text of DOI:10.1007/s13361-011-0170-4

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:1610Y1621
DOI: 10.1007/s13361-011-0170-4
RESEARCH ARTICLE
Tandem MS Analysis of Selenamide-Derivatized
Peptide Ions
Yun Zhang,1 Hao Zhang,2 Weidong Cui,2 Hao Chen1
1Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH,
45701, USA
2Department of Chemistry, Washington University in St. Louis, St. Louis, MO, USA
Abstract
Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)
phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in
high conversion yield. This paper reports the systematic investigation of MS/MS dissociation
behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and
electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit
tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions
preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment
ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from
protein digest as well as selective detection of thiol proteins from protein mixture using precursor
ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags
during CID, which is useful in sequencing peptides and locating cysteine residues. In the
negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage,
analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In
consideration of the convenience in preparing selenamide-derivatized peptides and the similarity
of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to
probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S
bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone
cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-
Washington mechanism proposed for depicting electron-based ion dissociation processes.
Key words: CID, ETD, Peptide ions, Selenamide, Thiol derivatization, Selective detection
tion), mass spectrometry (MS) [2, 3] has attracted significant
attention in the characterization of biological thiols. A
Introduction
number of elegant MS studies of thiols and disulfides of
proteins/peptides based on the novel ion chemistry have
been reported [4–9]. For MS analysis of thiol proteins/
peptides, the derivatization of thiol groups with a suitable
chemical reagent is often necessary for increasing thiol
stability and improving detection selectivity or for enrich-
ment and purification purposes. Furthermore, tandem MS
analysis (MS/MS) can benefit from the modification of
thiols. For instance, N,N-dimethyl-2-chloro-ethylamine [10]
was used to increase the charge state of cysteine-containing
toxin ions to meet the requirement for performing electron-
iological thiols such as glutathione and thiol proteins
are critical physiologic components found in animal
tissues and are involved in a plethora of important cellular
functions [1]. Due to the inherent sensitivity and chemical
specificity (e.g., molecular weight and structural informa-
B
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-011-0170-4) contains supplementary material, which
is available to authorized users.
Correspondence to: Hao Chen; e-mail: chenh2@ohio.edu
Received: 4 April 2011
Revised: 15 May 2011
Accepted: 15 May 2011
Published online: 9 June 2011

Y. Zhang, et al.: Peptide Ion Dissociation
1611
transfer dissociation (ETD) [11]. In addition, it has been
shown that thiol derivatization using (3-acrylamidopropyl)-
trimethyl ammonium chloride significantly improves the
percent fragmentation and sequence coverage for peptide
ions upon ETD [12]. Remarkably, quinone-modified cys-
teine was shown to facilitate site-selective cleavage by
photodissociation [13].
ions from backbone cleavage of peptides in the positive ion
mode. Interestingly, both tags can be removed upon CID in
the negative ion mode, which is similar to the known CID
behavior of disulfide bonds [28]. During ETD of derivatized
peptide ions, selenium tag loss is dominant while the
backbone cleavage is suppressed. Similar phenomenon was
also observed for alkali metal-containing peptide ions, which
can be explained by the Utah-Washington mechanism
proposed for ECD dissociation [22, 23].
Traditional thiol labeling strategies are mainly based on
nucleophilic substitution (e.g., using heptafluorobutyl chlor-
oformate [14] and iodoacetamide [15]), Michael-addition
with unsaturated C=C bonds (e.g., using acrylate [16] or
maleimide derivatives [17]), or thiol exchange reaction (e.g.,
using the Ellman’s reagent [18, 19]). However, these
protocols have some limitations such as low reaction
selectivity, long reaction time, or low conversion yield.
Recently we developed a new methodology to label bio-
logical thiols using selenamide reagents for MS analysis,
which involves the cleavage of Se–N bond by thiol to form a
new Se–S bond [20]. Our data show that the reaction is
highly selective, rapid, reversible and efficient. For instance,
among 20 natural amino acids, only cysteine is reactive
towards Se–N containing selenamide reagents such as
ebselen or N-(phenylseleno)phthalimide (NPSP), and the
reaction occurs in seconds. In the case of protein β-
lactoglobulin A containing one free cysteine residue, the
derivatization can be completed in 30 s with 100%
conversion yield. By adding reductant of dithiothreitol
(DTT), peptides derivatized by selenamide reagents can be
recovered. In order to better implement this new thiol
derivatization reaction for protein/peptide analysis by mass
spectrometry, it is therefore necessary to well understand the
tandem MS dissociation behavior of derivatized protein/
peptide ions, which is the motivation and focus of this study.
On the other hand, due to the significance of electron capture
dissociation (ECD) [21] and ETD [11] in top-down
proteomics research, their mechanisms arouse much atten-
tion and are still under debate [21–23]. One of the
approaches for mechanism elucidation is to introduce
various tags with different electron or proton affinities to
model peptides and then test the effects of the tags on the
dissociation [24–27]. Since the preparation of selenamide-
derivatized proteins/peptides is convenient as mentioned
above and that the formed Se-S bond is structurally similar
to the S–S bond, we also investigated ETD of the derivatized
peptide ions in effort to provide some new insight to the
electron-based ion dissociation mechanism.
Experimental
Chemicals
Peptides HCKFWW (MW 905.4 Da), NRCSQGSCWN
(MW 1153.4 Da), bovine pancreas insulin (MW
5733.5 Da), α-lactalbumin from bovine milk (type III,
calcium depleted, ≥85%, MW 14178 Da), β-lactoglobulin
A from bovine milk (MW 18369 Da), lysozyme from
chicken egg white (MW 14300 Da), pepsin from porcine
gastric mucosa (MW~35 KDa), NPSP, and tris(2-carbox-
yethyl)phosphine hydrochloride (TCEP) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Ebselen was
purchased from Calbiochem (Cincinnati, OH, USA). HPLC-
grade methanol from GFS Chemicals (Columbus, OH, USA)
and acetonitrile from Sigma-Aldrich (St. Louis, MO, USA)
were used and acetic acid was purchased from Fisher
Scientific (Pittsburgh, PA, USA). The deionized water used
for sample preparation was obtained using a Nanopure
Diamond Barnstead purification system (Barnstead Interna-
tional, Dubuque, IA, USA).
Preparation of Selenamide-Derivatized Peptides
Peptide thiols were derivatized by reaction with 5 ~10 time
excess amount of ebselen or NPSP in either methanol/water
(1:1 by volume) containing 1% acetic acid or acetonitrile/
water (1:1 by volume) containing 1% acetic acid at room
temperature.
The derivatized insulin chain B was obtained via reacting
the reduced insulin with the selenamide reagents. First TCEP
in 20 mM ammonium bicarbonate aqueous solution was
added to insulin in acetonitrile/water (1:1 by volume)
containing 0.1% trifluoroacetic acid in the molar ratio of
1:50 (protein/TCEP) for 3.5 h at room temperature. Then
Millipore-ZipTip Pipette Tips were used to remove TCEP
and ammonium bicarbonate via desalting. After that, excess
amount of ebselen (ebselen:protein=6:1) or NPSP (NPSP:
protein=11:1) were added to derivatize the reduced insulin.
In this study, we chose several free cysteine-containing
peptides as examples and systemically investigated their ion
dissociation pathways after derivatization by selenamide
reagents such as ebselen and NPSP, under CID (in both
positive and negative ion modes) and ETD conditions. For
the ebselen tag, it appears to be easily lost upon CID,
probably due to the presence of an adjacent amide group in
the tag, consistent with previous observation [20]. The
phenylselenenyl tag from NPSP derivatization can survive in
the CID process, leading to the formation of a series of b/y
Derivatization of Insulin Peptic Digest
and Protein Mixture
Digestion of insulin was performed by incubating insulin
and pepsin at a molar ratio of 50:1 in water containing 1%
acetic acid at 37 °C for 11 h. Then TCEP was added to the

1612
Y. Zhang, et al.: Peptide Ion Dissociation
peptic digested insulin in the molar ratio of 1:20 (protein:
TCEP) for several days at room temperature. After that,
ebselen (ebselen:protein=20:1) was added to derivatize the
insulin digest.
Ebselen (50 μM) was reacted with β-lactoglobulin A
(10 μM) in the presence of α-lactalbumin, lysozyme, and
insulin (10 μM for each) in methanol/water (1:1 by volume)
containing 1 % acetic acid for 30 s.
CID MS/MS of the Positive Ions of Selenamide-
Derivatized Peptides
Positive ion CID MS/MS of ebselen-derivatized peptide ions
was first carried out. Figures 1a–c show the CID MS/MS
mass spectra of doubly charged [HC*KFWW+2 H]2+ (m/z
591.0; see its structure in Figure 1a; the asterisk “*” denotes
one ebselen tag on the peptide cysteine group and such a
denotation is applicable throughout the text), doubly charged
[NRC*SQGSC*WN+2 H]2+ (m/z 852.8; structure shown in
Figure 1b), and triply charged [FVNQHLC*GSHLVEA-
LYLVC*GERGFFYTPKA+3 H]3+ (m/z 1317.0; structure
shown in Figure 1c), respectively. Upon CID, facile Se–S
bond cleavage occurs to m/z 591.0 (Figure 1a), leading to the
formation of singly and doubly charged ions of HCKFWW
(m/z 906.5 and 453.8) via the loss of one protonated ebselen
and one neutral ebselen, respectively. Indeed, the protonated
ebselen (m/z 276.1) was also observed in Figure 1a. This
process might be driven by the re-formation of the five-
membered ring of ebselen as assisted via the nucleophilic
attack of selenium by the adjacent amide nitrogen in the
ebselen tag. The result is consistent with our previous
observation in the case of ebselen-derivatized glutathione, a
tripeptide with one cysteine residue [20]. As illustrated in
Scheme 1, the cleavage of Se–S bond during CID takes
place in two pathways: one is to lose neutral ebselen
(pathway a, eq 1) and the other is to lose the protonated
ebselen (pathway b, eq 1). Also, in comparison to the Se–S
bond dissociation, the backbone cleavage to b/y ions is
negligible in this case and only a tiny peak of y4 ion was
seen. The facile Se–S bond cleavage appears to be a general
trend and was also observed during CID of the other two
peptides containing two ebselen tags (Figures 1b and c). In
Figure 1b, two major fragment ions of m/z 1429.4 and 715.2
were observed, corresponding to the singly and doubly charged
ions of NRCSQGSCWN with one ebselen tag, respectively,
arising from the losses of one protonated ebselen and one neutral
ebselen from [NRC*SQGSC*WN+2 H]2+ (m/z 852.8). The m/z
1429.4 was also further examined by MS/MS/MS, which shows
that the ebselen tag is either on its Cys-3 or on the Cys-8 residue
(shown in Figure S-2, Supporting Information). It indicates that
there is no regioselectivity for the dissociation of its precursor
ion of m/z 852.8 with regard to the ebselen tag loss. Another
fragment ion of m/z 1154.4 (Figure 1b), the protonated
NRCSQGSCWN originating from m/z 1429.4 dissociation by
further loss of the ebselen tag, was detected along with the
protonated ebselen and b/y fragment ions such as b4*, b5*, b6*,
b7*, and y9** with low abundances resulting from backbone
cleavage (again, the asterisk indicates one ebselen tag and the
double asterisks indicate two ebselen tags). In the case of insulin
chain B, m/z 1837.9 and 1225.7 representing the doubly and
triply charged ions of chain B with one ebselen tag were
produced upon CID, again, by losses of one protonated ebselen
and one neutral ebselen from the precursor ion of m/z 1317.0,
respectively. Also, the doubly and triply charged ions of the free
chain B were seen at m/z 1700.5 and 1134.3, respectively, as a
Mass Spectrometry
A LTQ-Orbitrap mass spectrometer (Thermo Scientific, San
Jose, CA, USA) with an electrospray source was used for
collecting CID and ETD data of derivatized peptide ions.
Peptide samples were directly ionized by ESI with the
injection flow rate of 3–5 μL/min. The spray voltage was 4–
4.5 kV. The automatic gain control (AGC) value was set at
1×106 (Orbitrap) and 3×104 (linear ion trap) with maximum
injection time of 500 ms (Orbitrap) and 200 ms (linear ion
trap). The high resolution product-ion spectra were acquired
at high mass resolving power (60 000 for ions of m/z 400).
The LTQ Orbitrap was externally calibrated using a standard
calibration mixture of caffeine, MRFA, and Ultramark 1621.
CID experiments were conducted in the linear ion trap with
helium as the collision gas. The normalized collision energy
used was 5%–35% with the activation time of 30 ms. The
isolation width was 2 Da. All ETD experiments were
conducted with fluoranthene radical anion as the electron-
transfer reagent in the linear ion trap. The ETD reaction time
was 60–120 ms. The precursor ion scan (PIS) was performed
using a hybrid triple quadrupole/linear ion trap mass
spectrometer (Q-trap 2000; Applied Biosystems/MDS
SCIEX, Concord, Canada) equipped with an electrospray
source. Samples were injected for ESI at the flow rate of 5
μL/min. The spray voltage was +5 kV. Curtain gas was 20.
Collision energy (CE) used was 50–80 eV. PIS was used to
screen the ebselen tag-containing precursor ions based on
the formation of the characteristic fragment ion of m/z 276
(i.e., the protonated ebselen) and N2 was used as the
collision gas.
Results and Discussion
Various biological thiols, such as peptide HCKFWW
containing one free cysteine, NRCSQGSCWN having
two free cysteines, and insulin chain B carrying two
cysteines, were chosen for examination in this study.
Following derivatization by ebselen or NPSP, the samples
were directly ionized by ESI, and it was found that the
number of tags in the main derivatization products agrees
with the number of the free cysteines of the peptide
substrates reacted (shown in Figure S-1, Supporting
Information). Then, the derivatized peptide ions were
mass-selected for tandem MS analysis.

Y. Zhang, et al.: Peptide Ion Dissociation
1613
y4
666.4
[HCKFWW+H]+
906.5
y5’
925.3
m/z 531.8
H C K F W W
(a)
m/z 591.0
(d)
y
2+
y1-NH3
100
2+
100
50
HCKFWW
b
CH2
CH
S
923.3
2
[HCKFWW+2H]2+
453.8
+2H
+2H
926.3
927.3
S
[ebselen+H]+
276.1
Se
667.5
668.4
669.4
O
Se
924.3
922.3
921.3
N
H
928.3
670.3
665 666 667 668 669 670 671 919 921 923 925 927 929 931
50
m/z
m/z
’
b2
-[ebselen+H]+
y1
’
b5
-ebselen
b4’-NH3 y4
m/z 591 .0
y5'
y2-NH3
y2
b5’-NH3
800
y3
y4
0
0
200
300
400
500
600
700
800
900
1000
200
300
400
500
600
700
900
(b)
(e)
100
[733.8-H2O+2H]2+
m/z 852.8
m/z 733.8
N R C S Q G S C W N
724.6
1429.4
y
2+
852.7
100
NRCSQGSCWN
2+
CH
CH
2
2
+2H
CH2
S
b
S
Se
CH2
S
Se
’’2+
S
Se
b9
O
O
+2H
’
b7
Se
N
H
N
H
’’
b9
’’
b8
50
50
0
’
b5
-[ebselen+H]+
y8”
[ebselen+H]+
275.9
y5’
y3’
-ebselen
715.2
’
b2
b4
y4’
’ y6’
b6
y2
1154.4
-ebselen
*
’
y7’
b
b3
6 b7
*
*
y9**
b5
*
b4
0
200
400
600
800
1000
y
1200
1400
400
600
800
1000 1200 1400 1600 1800 2000
m/z 1238.0
(c)
(f)
y24’’ 2+
3+
m/z 1317.0
1225.7
3+
100
100
50
0
F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K A
b27’’-H2O2+
CH
S
Se
CH
S
Se
2
2
b
b27’’3+
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
+3H
y25’’2+
CH
S
CH
S
2
2
+3H
-[ebselen+H]+
m/z 1317.0
-ebselen
Se
O
Se
O
y26’’2+
’’2+
b
2+
26
50
0
y23’
N
H
b6
b27’’2+
N
H
2+
y22’-NH3
b25’’2+
y28’’3+
2+
[B chain+2H]2+
1700.5
b14
b15
’
2+
y9
b16’
y15’-NH3
y11
[B chain+3H]3+
1134.3
2+
b5
’
y14’
y15’
y8
b15’
y
4 b4-NH3
b3
1837.9
b15
b6
b12*-NH3
b5
y10
y9
*
y11
y4
y8
400
600
800
1000
1200
1400
1600
1800
2000
400
600
800
1000
1200
m/z
1400
1600
1800
2000
m/z
Figure 1. Positive ion CID MS/MS mass spectra of (a) [ HC*KFWW+2 H]2+ (m/z 591.0); (b) [NRC*SQGSC*WN+2 H]2+
(m/z 852.8); (c) [FVNQHLC*GSHLVEALYLVC*GERGFFYTPKA+3 H]3+ (m/z 1317.0); (d) [HC’KFWW+2 H]2+ (m/z 531.8); (e)
[NRC’SQGSC’WN+2 H]2+ (m/z 733.8); (f) [FVNQHLC’GSHLVEALYLVC’GERGFFYTPKA+3 H]3+ (m/z 1238.0)
result of secondary dissociation of m/z 1837.9 and 1225.7 by
further loss of the remaining ebselen tag. These results clearly
suggest that ebselen-derivatized peptide cations preferentially
undergo Se–S bond cleavage during CID via losses of both
neutral and protonated ebselen (the exception of no protonated
ebselen observed in Figure 1c is probably due to the low mass
cut-off in the linear ion trap of the LTQ-Orbitrap used). Both the
formation of the characteristic fragment ion of m/z 276 and the
facile loss of neutral ebselen from the CID of derivatized thiol
peptide ions would be useful to identify thiol-containing
peptides from mixtures such as protein digests, using either
PIS or neutral loss scan (NLS), and the former application is
demonstrated below.
As a demonstration, insulin (its sequence is shown in the
inset of Figure 2b) was digested by pepsin and then reduced
by TCEP. The reduced protein digest mixture was derivat-
ized via the selective reaction with ebselen and then subject
to ESI-MS analysis. As illustrated in Figure 2a, the ESI-MS
spectrum shows that a series of ebselen-derivatized thiol
peptide ions [FVNQHLC*GSHL+3 H]3+ (m/z 510.8),
[HLC*GSHL +2 H]2+ (m/z 521.4), [LVC*GERGF+2 H]2+
(m/z 579.2), [QCCASVC*SL+2 H]2+ (m/z 596.0) (the exact
position of the ebselen tag in this case is uncertain),
[LVC*GERGFF+2 H]2+ (m/z 652.0), [YLVC*GERGF+
2 H]2+ (m/z 659.8), [YC*N+H]+ (m/z 674.3), [VC*SL+
H]+ (m/z 696.1), [QCC*ASVC*SL+2 H]2+ (m/z 733.5)
(the exact positions of the two ebselen tags are not
certain in this case), [FVNQHLC*GSHL+2 H]2+ (m/z
765.6), [LVC*GERGFFYT + 2 H]2+ (m/z 783.1),
[NYC*N +H]+ (m/z 788.2), [ASVC*SL+ H]+ (m/z
854.0), and [LVC*GERGFF+H]+ (m/z 1303.2) were
observed (listed in the Figure 2a inset). In addition,
[FVNQHLCGSHL + 2 H]2 +
,
[LVCGERGF + H]+ ,
[QCCASVCSL+H]+, [LVCGERGFF+H]+ were seen at
m/z 627.8, 880.5, 914.3, and 1027.1, respectively,
probably resulting from the incomplete derivatization
reaction by ebselen owing to the presence of the excess
amount of reductant TCEP (indeed, we found that, like
DTT, TCEP can reduce the ebselen-derivatized peptides
such as glutathione in a separate experiment). Further-

1614
Y. Zhang, et al.: Peptide Ion Dissociation
O
2+
HCKFWW
CH2
a
b
[HCKFWW+2H]2+
+
N
Se
ebselen
+2H
(eq. 1)
S
H
N
Se
[HCKFWW+H]++ [ebselen+H]+
O
-
HCKFWW
CH2
S
Se
-H
[HCKFWW-H]-+ ebselen
(eq. 2)
H
N
O
Scheme 1. CID MS/MS processes for [HC*KFWW+2 H]2+ (eq 1) and [HC*KFWW – H]– (eq 2)
more, [ALY+H]+, [GIVE+H]+, [VEAL+H]+, [FVNQ+
H]+, [YQLE+H]+, [YTPKA+H]+, and [QLENY+H]+
were detected at m/z 366.1, 417.2, 431.3, 507.4, 552.2,
579.2, and 666.4, respectively (shown in the inset of
Figure 2a). These ions correspond to seven additional
peptides, which do not have free cysteine residues and, as
a result, no corresponding ebselen-derivatization ions
were detected for these peptides. Thus, in this insulin
digest, a total of 18 peptides were observed (listed in the
Figure 2b inset), covering 100% of the sequence of
insulin. Meanwhile, [2(ebselen)+H]+ was also detected at
m/z 550.9. Due to the multi-component nature of the
sample, the ESI-MS spectrum in Figure 2a has multiple
peaks and appears complicated. Using the Q-trap instru-
ment, PIS was employed to screen those peptides
containing cysteine residues. By monitoring the product
ion of m/z 276, all of the ebselen-derivatized peptide ions
mentioned above were selectively detected corresponding
to 11 thiol peptides, as shown in Figure 2b (inset also
shows the list of detected thiol peptides). This experiment
provides a simple and rapid method to selectively
identify thiol peptides in complex protein digest mix-
tures. This method also complements our previous
method for selective identification of disulfide bond-
containing peptides in protein digests based on their
response to electrochemical reduction [29]. Furthermore,
this selective strategy is also applicable to thiol proteins.
In another experiment, a protein mixture containing β-
lactoglobulin A, α-lactalbumin, lysozyme, and insulin
was derivatized via the selective reaction with ebselen and then
analyzed by ESI-MS. As illustrated in Figure 3a, the ESI-MS
spectrum shows the multiply charged ions of α-lactalbumin,
lysozyme, and insulin, together with one ebselen-derivatized β-
lactoglobulin A ions. PIS was then applied to screen the
thiol protein ions. As shown in Figure 3b, the ebselen-
derivatized β-lactoglobulin A ions were selectively
detected by monitoring the product ion of m/z 276. Thus,
it can be seen that the ebselen derivatization in con-
junction with PIS is powerful in the analysis of both thiol
peptides and proteins in mixtures.
In stark contrast to ebselen tagging, peptide ions with
phenylselenenyl tags arising from NPSP derivatization
display drastically different CID dissociation behaviors, in
which the tags are preserved and consecutive backbone
cleavages occur (eq 1, Scheme 2). This is probably due to
the lack of intramolecular nucleophilic attack of selenenyl
sulfide bond by adjacent amide nitrogen of the selenium
tag as in the case of ebselen. As shown in Figure 1d,
extensive b and y ions (b2', b4'-NH3, b5', b5'-NH3, y1, y1-
NH3, y2, y2-NH3, y3, y4, y5') were seen (the single prime
indicates one phenylselenenyl tag). Again, similar phe-
nomena were observed for CID of the NRCSQGSCWN
ion with two phenylselenenyl tags, [NRCSQ'GSC'WN+
2 H]2+ (m/z 733.8, Figure 1e), and of the insulin chain B
ion with two phenylselenenyl tags, [FVNQHLC'GSHL-
VEALYLVC'GERGFFYTPKA+3 H]3+ (m/z 1238.0,
Figure 1f). For the former peptide ion, mainly b2, b3',
b4', b5', b6', b7', b8'', b9'', b9''2+, y2, y3', y4', y5', y6', y7',
and y8'' were observed (Figure 1e; the double prime
indicates two phenylselenenyl tags in the fragment ions).
For the latter, b3, b4-NH3, b5, b6, b14'2+, b15', b15'2+, b16'2+,
b25''2+, b26''2+, b27''2+, b27''-H2O2+, b27''3+, y4, y8, y9, y11, y14',
y15', y15'-NH3, y22'-NH32+, y23'2+, y24''2+, y25''2+, y26''2+, and
y28''3+were detected (Figure 1f). It turns out that the phenyl-
selenenyl tag could facilitate peptide sequencing and be
valuable in pinpointing the location of cysteine residues
of peptides. For example, based on the distinct selenium
isotope distribution of y5' which is absent for y4
(Figure 1d inset), it is clear that y5' contains the tag and
thus a cysteine residue while y4 does not. This result is in
agreement with the peptide structure (the cysteine is
located in the second residue of the peptide HCKFWW)
and also emphasizes the high selectivity of the NPSP
derivatization toward cysteine. The derivatization selec-
tivity was further confirmed by the appearance of frag-
ment ion pairs of b2/b3' and b7'/b8'' in the case of peptide
NRCSQGSCWN (having Cys-3 and Cys-8) and the
generation of the fragment ion pairs of y11/y14' and y23'2+/
y24''2+ in the case of insulin chain B (having Cys-7 and
Cys-19). In particular, the pair y11/y14' reveals that one of

Y. Zhang, et al.: Peptide Ion Dissociation
1615
Insulin digest, ESI-MS
(a)
9
1. m/z 366.1 [ALY+H]+
17. m/z 733.5 [QCC*ASVC*SL+2H]2+
3.0e6
2. m/z 417.2 [GIVE+H]+
18. m/z 765.6 [FVNQHLC*GSHL+2H]2+
19. m/z 783.1 [LVC*GERGFFYT+2H]2+
20. m/z 788.2 [NYC*N+H]+
3. m/z 431.3 [VEAL+H]+
18
4. m/z 507.4 [FVNQ+H]+
5. m/z 510.8 [FVNQHLC*GSHL+3H]3+
6. m/z 521.4 [HLC*GSHL+2H]2+
7. m/z 550.9 [2(ebselen)+H]+
8. m/z 552.2 [YQLE+H]+
21. m/z 854.0 [ASVC*SL+H]+
22. m/z 880.5 [LVCGERGF+H]+
23. m/z 914.3 [QCCASVCSL+H]+
24. m/z 1027. 1 [LVCGERGFF+H]+
25. m/z 1303. 2 [LVC*GERGFF+H]+
2.0e6
9. m/z 579.2 [YTPKA+H]+
[LVC*GERGF+2H]2+
10. m/z 596.0 [QCCASVC*SL+2H]2+
11. m/z 627.8 [FVNQHLCGSHL+2H]2+
12. m/z 652.0 [LVC*GERGFF+2H]2+
13. m/z 659.8 [YLVC*GERGF+2H]2+
14. m/z 666.4 [QLENY+H]+
15. m/z 674.3 [YC*N+H]+
12
8
6
5
19
1
3
16. m/z 696.1 [VC*SL+H]+
* = ebselen tag
2
1.0e6
4
11
14
15
20
21
22
24
16
7
17
10
13
25
23
300
350
400
450
500
550
600
650
700
750
800
850
900
950 1000 1050 1100 1150 1200 1250 1300 1350
Insulin digest, Precursor ion scan m/z 276
18
765.5
12
651.9
(b)
Insulin
SS
GIVEQCCASVCSLYQLENYCN
Chain A
Chain B
5.0e5
4.0e5
3.0e5
2.0e5
1.0e5
S
S
S
S
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
7
550.9
A list of detected peptides:
Cys-containing
FVNQHLCGSHL
HLCGSHL
LVCGERGF
LVCGERGFF
LVCGERGFFYT
QCCASVCSL
YLVCGERGF
YCN
no cys-containing
19
783.3
ALY
GIVE
VEAL
FVNQ
YQLE
YTPKA
QLENY
6
521.8
5
NYCN
VCSL
ASVCSL
511.1
9
20
787.7
21
854.0
17
733.1
578.5
15
674.1
16
13
660.0
695.7
10
595.5
25
1303.1
300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350
m/z
Figure 2. (a) ESI-MS mass spectrum showing the reaction of the reduced peptic digested insulin with ebselen. The inset
shows the list of all the peptide peaks; (b) PIS based on the monitoring of the characteristic fragment ion of m/z 276 upon CID
shows the selective detection of thiol peptides in the reduced peptic digested insulin. The insulin sequence and a list of
detected cysteine-containing and non-cysteine containing peptides are shown in the inset
the two cysteines is located between the 17th and 19th
residues of the chain B.
[32]. It is known that S–S undergoes marginal cleavage
under positive ion CID except with high energy or with
metal ions [33]. But S–S undergoes facile cleavage under
negative ion CID [3, 34]. Proton abstraction from the Cα and
Cβ of the cysteine residue causes diverse fragmentation
reactions including the cleavage of disulfide bonds. Persul-
fides, thioaldehydes, and dehydroalanine residues at the
cysteine position are generated upon negative ion CID [35].
Therefore we also investigated the dissociation behavior of
the selenamide-derivatized peptide ions containing Se–S
bonds under negative ion CID conditions. The basic
CID MS/MS of the Negative Ions of Selenamide-
Derivatized Peptides
Selenium and sulfur, two elements in the Group VIB, exhibit
many expected similarities in chemical properties [30, 31].
Indeed, they have similar electronegativities of 2.55 and 2.58
(Pauling scale), respectively. The Se–S bond is also close to
the S–S bond, both of which can be electrolytically reduced

1616
Y. Zhang, et al.: Peptide Ion Dissociation
Figure 3. (a) ESI-MS mass spectrum showing a mixture of β-lactoglobulin A, α-lactalbumin, lysozyme and insulin after reaction
with ebselen; (b) precursor ion scanning based on the monitoring of the characteristic fragment ion of m/z 276 upon CID shows
the selective detection of thiol containing protein β-lactoglobulin A in a protein mixture
cleavages of Se–S bond in ebselen- and NPSP-derivatized
peptide ions are outlined in eq 2 (Schemes 1 and 2). For the
fragmentation of ebselen-derivatized peptide anions, it is
similar to that of their cationic counterparts and the loss of
neutral ebselen is the major dissociation pathway (eq 2,
Scheme 1). As shown in the CID MS/MS mass spectrum of
[HC*KFWW – H]– (m/z 1179.0, Figure 4a), m/z 904.5
corresponding to [HCKFWW – H]– was generated by loss of
one ebselen. In the case of CID of the NPSP-derivatized
peptide anion, the elimination of the proton from α and β
carbons of the cysteine residue gives rise to the thioaldehyde
and dehydroalanine moieties, respectively (eq 2, Scheme 2).
These processes can be seen in the CID MS/MS mass spectrum
of [HC'KFWW – H]– (m/z 1060.0, Figure 4b), which results in
intense fragment ions of m/z 902.3 and 870.4. It does confirm
that Se–S cleavage occurs in negative ion CID as expected. The
negative ion CID might be useful in the analysis of acidic
peptides as those peptides prefer to form anions.
ETD MS/MS of Selenamide-Derivatized Peptide
Ions
Both ECD [21] and ETD [11] are powerful dissociation
methods which often give rise to extensive backbone
cleavages. In ECD experiments, low-energy electrons are
reacted with peptide cations in the ICR cell located in the
center of the magnetic field of a Fourier-Transfer Ion
cyclotron resonance mass spectrometer (FT-ICR-MS) [21].

Y. Zhang, et al.: Peptide Ion Dissociation
1617
2+
HCKFWW
CH2
S
b, y ions
(eq. 1)
+2H
Se
-
HCKFWW
HCKFWW
a
b
+ PhSeH
CH2
CH
S
-H
S
Se
-H
(eq. 2)
HCKFWW
CH2
+ PhSeSH
-H
Scheme 2. CID MS/MS processes for [HC'KFWW+2 H]2+ (eq 1) and [HC'KFWW – H]– (eq 2)
ETD utilizes ion/ion reactions [11, 36, 37] to transfer
electron to peptide ions for dissociation but is regarded to
have similar mechanism to ECD. An early proposed
mechanism for ECD (also known as the Cornell mechanism
[21]) starts with electron localization at positively charged
ammonium or guanidinium groups of a peptide that
generates a hypervalent center [28], which transfers a
hydrogen atom H. to a carbonyl oxygen of an amide group
to form an aminoketal radical intermediate. The aminoketal
radical intermediate subsequently undergoes the cleavage of
the N– Cα bond to form c/z ions [38]. It has been previously
shown that disulfide bonds can be preferentially cleaved by
ECD because the S–S bond has a higher affinity for the H.
atom compared with the amide carbonyl [28]. However, the
mechanism of ECD and ETD fragmentations are still not
thoroughly understood [21–23, 39]. Even though the Cornell
(a)
m/z1179.0
904.5
100
HCKFWW
CH
S
Se
2
HCKFWW
CH
-H
1178.9
2
-H
O
SH
N
H
50
-ebselen
0
400
500
600
700
800
900
1000
1100
1200
(b)
m/z1060.0
902.3
100
50
0
HCKFWW
HCKFWW
CH
S
Se
2
CH
S
-H
-H
1060.2
HCKFWW
-PhSeH
-H
CH
2
870.4
900
300
400
500
600
700
m/z
800
1000
1100
Figure 4. CID MS/MS mass spectra of the negative ion of (a) [HC*KFWW – H]– (m/z 1179.0) and (b) [HC'KFWW – H]– (m/z 1060.0)

1618
Y. Zhang, et al.: Peptide Ion Dissociation
mechanism provided a reasonable picture for ECD, some
backbone fragmentations were not easily explained [24, 40].
An alternative mechanism, now known as the Utah-
Washington mechanism [22, 23], has been proposed to
explain the origin of c and z fragment ions. In this theory,
direct electron attachment to Coulomb-stabilized amide π*
orbitals makes the amide group an exceptionally strong base
with a proton affinity (PA) in the range of 1100–1400 kJ/
mol [41]. The amide anion radical is able to abstract a proton
to result in an intermediate identical to the aminoketyl cation
radical proposed in the Cornell mechanism and can undergo
the same N–Cα bond cleavage. Recently, one of the
powerful approaches for the ECD/ETD mechanistic eluci-
dation is to introduce various tags with different electron or
proton affinities to model peptides and to test the effect of
the tags on the dissociation upon electron capture/transfer
[24–27]. In consideration of convenient preparation of
selenamide-derivatized proteins/peptides and the structural
similarity of the resulting Se–S bond to S–S bond, electron-
based dissociation behaviors of the derivatized peptide ions
were also examined, with the purpose of providing some
insights into the mechanism of the electron-based dissociation.
Figure 5a and d show the ETD MS/MS mass spectra of
[HC*KFWW+2 H]2+ and [HC'KFWW+2 H]2+, respec-
tively. In both cases, preferential cleavage of selenium tag
takes place, giving rise to the main fragment ion of m/z
906.5, the protonated peptide ion without the tag. Also, m/z
1063.3 corresponding to the radical cation [HC' KFWW+
2 H]+▪ as a result of one electron capture is shown in
Figure 5d. Few c ions with very low abundances from
backbone cleavage were observed. The ETD process for the
doubly charged ion of HCKFWW with one selenium tag is
illustrated in eq 1, Scheme 3. The cleavage of the Se–S
bond can be expedited by direct electron-capture as
stabilized by the Coulomb effect (by the positively charged
arginine or terminal amino of the peptide) followed with
bond cleavage, leading to the loss of radical RSe▪ and
thiolate anion. Subsequent proton transfer occurring to the
thiolate anion forms singly charged HCKFWW. This
proposed mechanism follows the Utah-Washington pattern
ETD m/z 591.0
ETD m/z 531.8
(a)
(d)
[HCKFWW+H]+
906.5
[HCKFWW+H]+
906.5
2+
HCKFWW
2+
100
+
HCKFWW
CH2
HCKFWW
CH2
100
CH
S
2
+2H
+2H
S
+2H
S
531.8
O
Se
Se
Se
N
H
Se
50
-
Se
50
O
591.3
600
N
H
-
-H2O
c5’
1063.3
-H2O
c5’
c4’
c3’
600
c3’
0
0
200
400
800
1000
1200
1400
200
300
400
500
700
800
900
1000
(b)
ETD m/z 852.8
(e)
ETD m/z 733.8
2+
NRCSQGSCWN
1429.4
2+
733.6
NRCSQGSCWN
100
CH2
CH2
100
1310.4
CH2
S
CH2
S
+2H
S
Se
S
Se
O
O
+2H
Se
Se
N
N
H
+
H
Se
NRCSQGSCWN
Se
O
CH
S
CH
S
Se
852.1
2
2
+
-
NRCSQGSCWN
N
H
-
+2H
CH2
CH2
Se
50
+2H
S
Se
50
S
Se
O
O
NRCSQGSCWN
NRCSQGSCWN
N
H
N
H
CH2
S
CH2
SH
CH
S
CH
SH
2
2
-NH3
1152.5
1703.6
1600 1800
1152.5
1200
1466.3
1400
’
’
b5
b7
0
0
400
600
800
1000
1400
2000
200
400
600
800
1000
1200
(c)
(f)
ETD m/z 1317.0
ETD m/z 1238.0
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
3+
1778.5
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
3+
1838.0
100
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
2+
100
CH2
S
Se
CH2
S
Se
CH2
S
Se
CH2
S
CH2
S
CH2
S
+3H
+3H
+3H
Se
Se
O
Se
O
Se
Se
O
1238.0
N
H
N
H
-
N
-
50
H
2+
50
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
1856.4
1800
1316.9
CH
S
Se
CH
S
Se
2
2
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
+3H
CH
S
CH
2
+ H
2
O
O
SH
1975.5
N
1699.9
1600
N
H
H
0
0
400
600
800
1000
1200
1400
2000
200 400 600 800 1000 1200 1400 1600 1800 2000
m/z
m/z
Figure 5. ETD MS/MS mass spectra of (a) [HC*KFWW+2 H]2+ (m/z 591.0); (b) [NRC*SQGSC*WN+2 H]2+ (m/z 852.8); (c)
[FVNQHLC*GSHLVEALYLVC*GERGFFYTPKA+3 H]3+ (m/z 1317.0); (d) [HC’KFWW+2 H]2+ (m/z 531.8); (e) [NRC’SQGSC’WN+
2 H]2+ (m/z 733.8); (f) [FVNQHLC'GSHLVEALYLVC'GERGFFYTPKA+3 H]3+ (m/z 1238.0)

Y. Zhang, et al.: Peptide Ion Dissociation
1619
2+
HCKFWW
CH2
HCKFWW
CH2
HCKFWW
+e-
+2H
+2H
S
S
CH2
+H
SH
+RSe
+RSe
Se
Se
R
R
2+
HCKFWW
CH2
HCKFWW
CH2
HCKFWW
+e-
+Na+K
CH2
SNa
S
Se
S
+K
+Na+K
Se
R
R
O
N
H
R= Ph or
Scheme 3. Dissociation processes for the doubly charged HCKFWW carrying two protons and one selenium tag (eq 1) and for
the doubly charged HCKFWW carrying one sodium, one potassium, and one selenium tag (eq 2) upon ETD
and was further confirmed by ETD of the corresponding
alkali metal-containing peptide ion. Figure 6 shows ETD
MS/MS mass spectrum of [HC'KFWW+Na+K]2+ (m/z
562.2), and preferential cleavage of the phenylselenenyl
tag was also observed to take place to form the fragment
ion of m/z 967.3 ([HCKFWW+Na+K – H]+) (see the
proposed mechanism in eq 2, Scheme 3). Also, m/z 1124.3
corresponding to [HC'KFWW+Na+K]+▪ is shown in
Figure 6 as the charge reduced species. We also tested
ETD of [C*QDSETRTFY+2Na]2+ (m/z 784.8) and [C'QD-
SETRTFY+2Na]2+ (m/z 725.3) and the losses of the
selenium tags were observed as well (Figure S–3, Support-
ing Information). Again, as shown in Figure 5b and e, the
NRCSQGSCWN peptide ions with two ebselen or phenyl-
selenenyl tags also display similar ETD mass spectra. No c
or z ions were observed, instead only few b/y ions with low
abundances were detected. In both cases (Figure 5b and e),
preferential cleavage of one selenium tag occurs predom-
inantly, generating the main fragment ions of m/z 1429.4
and 1310.4, respectively. The ETD mass spectra of
[FVNQHLC*GSHLVEALYLVC*GERGFFYTPKA+3 H]3+
and [FVNQHLC'GSHLVEALYLVC'GERGFFYTPKA+
3 H]3+ were shown in Figure 5c and f, respectively.
Likewise, there are no c or z ions observed, either. The
cleavage of one ebselen tag occurs to either Cys-7 or Cys-19,
giving rise to the fragment ion of m/z 1838.0 (shown in
Figure 5c). Because of the presence of two selenenyl sulfide
bonds in selenamide-derivatized NRCSQGSCWN and insulin
chain B, the electron transfer to the tag completely suppresses
the backbone cleavage resulting in no formation of c/z ions.
These results indicate that the selenium tags can efficiently
quench the formation of c/z ions in the ETD of peptide ions,
and the mechanism can be accounted for by the Utah-
Washington mechanism. On the other hand, although the
562.2
100
ETD m/z 562.2
HCKFWW
CH2
+Na+K
S
2
HCKFWW
CH2
+Na+K
S
Se
Se
50
1124.3
Se
[HCKFWW+Na+K-H]+
967.3
-
0
200
400
600
800
1000
1200
1400
m/z
Figure 6. ETD MS/MS mass spectrum of [HC'KFWW+Na+K]2+(m/z 562.2)

1620
Y. Zhang, et al.: Peptide Ion Dissociation
prohibition of backbone cleavage occurs to derivatized
peptide ions, extensive c/z ions can still be seen for ECD
of selenamide-derivatized protein ions, as we will report
in due course.
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Conclusions
In conclusion, unimolecular ion dissociation behaviors of
selenamide-labeled thiol peptide ions upon CID and ETD
were investigated. It is evident that derivatized peptide
cations undergo tag-dependent CID dissociation pathways.
Ebselen-derivatized peptide cations display the unique frag-
ment ion of m/z 276 upon dissociation, which is useful for
selective identification of thiol peptides and proteins in
mixture. The robust phenylselenenyl tag is useful in peptide
sequencing and locating of cysteine residues in peptides. In
the negative ion mode CID, both types of tags are
preferentially lost via the Se–S cleavage, similar to S–S
bond. In addition, the preferential cleavage of Se–S bond
occurs over the formation of c/z ions during ETD activation
to both protonated and alkaliated peptides, following the
Utah-Washington mechanism. Given the significance of
thiol residues in proteins/peptides, the selective derivatiza-
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revealed in this study would be useful in the proteomics
research.
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Acknowledgments
The authors gratefully acknowledge support of this work by
NSF (CHE-0911160). Y.Z. and H.C. are thankful to
Professor Michael L. Gross for the access to the NIH/NCRR
Mass Spectrometry Resources at Washington University in
St. Louis (grant number 2P41RR000954).
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