Gas-phase peptide sulfinyl radical ions: Formation and unimolecular dissociation

Article abstract of DOI:10.1007/s13361-012-0465-0

A variety of peptide sulfinyl radical (RSO) ions with a well-defined radical site at the cysteine side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and investigated via collision-induced dissociation (CID). The radical ion formation was based on AP reactions between oxidative radicals and peptide ions containing single inter-chain disulfide bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical induced reactions allowed large flexibility in forming peptide radical ions independent of ion polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20 peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition between radical- and charge-directed fragmentation pathways was largely affected by the presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e., singly protonated, containing basic amino acid residues), loss of 62 Da (CH₂SO), a radical-initiated dissociation channel, was dominant. For systems with mobile protons, this channel was suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge was found to significantly alter the radical-initiated dissociation channels, which might be related to the difference in stability of the product ions in different ion charge polarities. American Society for Mass Spectrometry, 2012.

Full text of DOI:10.1007/s13361-012-0465-0

B American Society for Mass Spectrometry, 2012
J. Am. Soc. Mass Spectrom. (2012)
DOI: 10.1007/s13361-012-0465-0
RESEARCH ARTICLE
Gas-Phase Peptide Sulfinyl Radical Ions:
Formation and Unimolecular Dissociation
Lei Tan, Yu Xia
Department of Chemistry, Purdue University, West Lafayette, IN 47907-2084, USA
Abstract
A variety of peptide sulfinyl radical (RSO•) ions with a well-defined radical site at the cysteine
side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and
investigated via collision-induced dissociation (CID). The radical ion formation was based on AP
reactions between oxidative radicals and peptide ions containing single inter-chain disulfide
bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical
induced reactions allowed large flexibility in forming peptide radical ions independent of ion
polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20
peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy
collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition
between radical- and charge-directed fragmentation pathways was largely affected by the
presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e.,
singly protonated, containing basic amino acid residues), loss of 62 Da (CH2SO), a radical-
initiated dissociation channel, was dominant. For systems with mobile protons, this channel was
suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge
was found to significantly alter the radical-initiated dissociation channels, which might be related
to the difference in stability of the product ions in different ion charge polarities.
Key words: Peptide radical ion, Sulfinyl radical, Disulfide bond, Collision-induced dissociation,
Tandem mass spectrometry
to analyze the stable reaction products of biomolecules after
Introduction
exposure to radical attack in solution [10–12]. The distonic
ion approach pioneered by Kenttämaa and co-workers
allows exploration of radical attack on biomolecules via
gas-phase ion/molecule reactions [13]. In those studies,
distonic ions (ions with separated charge and radical site)
were used as surrogates of radicals to react with neutral
amino acids or dipeptides, and several classes of reaction
channels were identified [14, 15]. Another MS approach is
to directly form and study peptide/protein radical ions in the
gas-phase. Although electron ionization (EI) has been
widely used to produce radical cations of small organic
compounds [16], it is difficult to be applied to biomolecules,
which have low vapor pressure and easily decompose upon
heating. Developing versatile approaches of generating gas-
phase biomolecule radical ions remains an active research
area. Electron transfer or electron capture processes give rise
rotein radicals, although typically existing as transient
species, carry out important roles in biological systems
P
[1]. Several classes of enzymes utilize radicals as active sites
to control their catalytic functions [2–5]. In addition, protein
radicals are often produced as intermediates during oxidative
damage of proteins induced by reactive oxygen species [6–
9]. Investigating the chemical properties of protein radicals
is highly desirable in order to understand the associated
biological events. Mass spectrometry (MS) has been utilized
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-012-0465-0) contains supplementary material, which
is available to authorized users.
Correspondence to: Yu Xia; e-mail: yxia@purdue.edu
Received: 5 June 2012
Revised: 27 July 2012
Accepted: 31 July 2012

L. Tan and Y. Xia: Peptide Sulfinyl Radical Ions
to hydrogen rich radical cations, in which the peptide contains
more hydrogen(s) than the corresponding even electron species
[17–20]. Dissociation of the hydrogen rich radical cations often
gives rise to c and z• ions, along with side-chain losses, through
which they can convert quickly to hydrogen deficient radical
ions [19–21]. Hydrogen deficient cations can be generated by a
variety of methods: laser ablation followed by ultraviolet (UV)
photoionization [22, 23]; collision-induced dissociation (CID)
of metal-ligand-peptide complexes [24–26]; CID of peptide
derivatives with labile bonds such as S-nitrosylation [27, 28],
serine/homoserine nitrate esters [29], peroxycarbamates [30],
2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO) [31, 32], and
4,4'-azobis(4-cyanopentanoic acid) (Vazo 68) [33]; UV photol-
ysis of iodinated tyrosine containing peptides [34]; or non-
covalent complexes with photolabile precursor [35]; electron-
induced dissociation of multiply charged ions [36, 37].
Hydrogen deficient radical anions can be formed by electron
detachment [38] or photodetachment [39] from multiply
deprotonated molecules, CID of peptide–metal complexes [40,
41], and photodissociation of iodinated peptide [42].
By introducing a radical site to a peptide ion, the gas-phase
ion chemistry can be drastically altered from the even-electron
counterpart. Several groups have utilized both experimental
approaches (i.e., ion/molecule reactions [14, 15, 27] and ion
spectroscopy [43–45]) and theoretical calculations [21, 46–49]
to investigate the structures of amino acid or small peptide
radical ions. Radical ion chemistry, such as intramolecular
radical migration [27, 50] and competition between charge- and
radical-directed dissociation upon collisional activation [49–51]
have also been explored with different chemical systems. The
capability of controlling the radical site upon its formation is
highly desirable in studying the fundamental aspects as
mentioned above.
Small organic sulfinyl radicals, such as HSO• and CH3SO•,
are important products for oxidation of pollutants like H2S and
CH3SH, as well as photolysis of sulfoxide, and thus play an
important role in the atmospheric sulfur cycles [52–54]. Protein
sulfinyl radicals have been detected by electron spin resonance
spectroscopy for glycyl-radical enzyme system upon its expo-
sure to molecular oxygen [55, 56]. It was suggested that the
sulfinyl radical species might participate in adjusting the glycyl/
thiyl radical equilibrium and reactivity of radical enzyme [55,
57]. Due to the transient nature of radical species in solution,
very little has been explored on the chemical property of protein
or peptide sulfinyl radicals. In previous studies, we observed that
disulfide bonds within peptides could be cleaved when the
peptide nanoelectrospray plume was allowed to interact with the
after-glow of a helium plasma in air, resulting in the formation of
sulfinyl radical (−SO•) and sulfhydryl (−SH) at the cleavage site
[58, 59]. Atmospheric pressure (AP) reactions induced by
reactive radicals were suggested to be responsible for the
oxidative cleavage of the disulfide bond. The radical induced
reactions allowed the formation of a series of peptide radical ions
(noted as [M + nH + OH]n•+) containing sulfinyl radical when
using intra-chain disulfide linked peptides. However, the
location of the radical site was ambiguous given that the sulfinyl
radical could be formed at either cysteine residue. In this study,
AP reactions between oxidative radicals and peptides containing
single inter-chain disulfide bonds or free cysteine residues were
developed to form peptide sulfinyl radical cations or anions with
known radical location. With this capability, the gas-phase
unimolecular dissociation of more than 20 peptide sulfinyl
radical ions was investigated via CID. In this study, we intend to
shed light on how peptide sequence, charge states, and charge
polarity would affect the fragmentation behavior of peptide
sulfinyl radical ions, and gain insight into the competition
between charge- and radical-directed dissociation in peptide
radical ions.
Experimental
Materials
Peptides used in this study are listed in Tables 1 and 2. Most
peptides with single inter-chain disulfide bonds were produced
either from enzymatic digestion of peptides with an intra-chain
disulfide bond or from oxidation of single cysteine containing
peptide to form the disulfide linked dimer. Peptides
CLPTRHMAC (disulfide bond: 1–9), CGNKRTRGC (disul-
fide bond: 1–9), CSRNLIDC (disulfide bond: 1–8), CNGRC-
NH2 (disulfide bond: 1–5), CIELLQARC (disulfide bond: 1–
9), CYAAPLKPAKSC (disulfide bond: 1–12), and
AGCKNFFWKTFTSC (disulfide bond: 3–14) were pur-
chased from Anaspec (San Jose, CA, USA). These peptides
were subjected to trypsin digestion with an enzyme/substrate
ratio of 1:50, and incubated at 37 °C in 50 mM ammonium
bicarbonate buffer for 2 h. The tryptic digestion of these
peptides gave rise to peptides 1–7 as listed in Table 1,
respectively. Peptide 8 was formed from pepsin digestion of
peptide CTTHWGFTLC (disulfide bond 1–10, Anaspec). The
pepsin digestion was performed in 5 % formic acid with an
enzyme/substrate ratio of 1:50 at 37 °C for 4 h. Peptide 9 was
Table 1. List of Inter-Chain Disulfide Peptides for the Formation of Peptide
Sulfinyl Radical Ions. Each Chain within the Peptide was Denoted with “A”
or “B”
Label
Sequence
CLPTR
HMAC
CGNKR
GC
Label
Sequence
CYAPPLKPAK
A
B
A
B
1
6
SC
AGCK
TFTSC
CTTHWGF
TLC
A
B
A
B
2
3
4
5
7
8
CSR
A
B
A
B
NLIDC
CGAILR
CGAILR
CNGR
C-NH2
A
B
A
B
9
CIELLQAR
C
γECG
γECG
A
B
A
B
10

L. Tan and Y. Xia: Peptide Sulfinyl Radical Ions
Table 2. List of Peptides Containing Free Cysteine Residues for the
Formation of Peptide Sulfinyl Radical Ions
Results and Discussion
Nomenclature
Label
Sequence
Label
Sequence
‘M’ was used to describe an even electron peptide species in its
original state, with no addition or subtraction of protons. “A”
and “B” were used to denote the different chains for inter-chain
disulfide peptides based on homolytic cleavage of the disulfide
bond (without any extra atom attached to the sulfur after
11
12
13
14
CLPTR
CAR
RGDC
GCGK
15
16
17
18
RGGALC
RGCALG
RVCIHPF
CFRHDSGY
SH
SO•
cleavage). Superscripts,
and
to the left of “A” or “B”
formed upon oxidation of peptide CGAILR (CPC Scien-
tific, Sunnyvale, CA, USA) by adding 10 μL of 1 %
hydrogen peroxide to 1 mL of 10 μM peptide in 50/50
MeOH/H2O solution, and stirring for 5 h. Peptide 10,
oxidized glutathione, was bought from Sigma-Aldrich (St.
Louis, MO). Peptide 11 was generated by reduction of
disulfide bond in peptide CD 154 (CLPTRHMAC, 1–9
disulfide bond, Anaspec), followed by trypsin digestion.
Reduction of disulfide bond was achieved by mixing
10 μL peptide solution (1 mg/mL in water) with 10 times
molar excess of 1 mg/mL dithiothreitol solution (DTT,
Sigma-Aldrich). The pH of solution was adjusted to 8 by
adding 1 % ammonium hydroxide and the final mixture
was allowed to react for 30 min at room temperature.
Peptides 12 and 14 were synthesized by CPC Scientific.
Peptide 13 and 18 were purchased from Anaspec. Peptides
15–17 were synthesized by SynBioSci (Livermore, CA,
USA). Working solutions for nanoESI were prepared to a
final concentration of 10 μM of a peptide in 50/49/1
MeOH/H2O/HOAc (vol/vol/vol).
chain symbols indicated the formation of sulfhydryl (−SH) and
sulfinyl radical (−SO•) at the cysteinyl sulfur, respectively. In
cases where sequences of the peptides were written out, SO•
C
was used to emphasize the existence of sulfinyl radical within
the peptide ions. For fragments derived from peptide sulfinyl
ions, superscript, SO• again was used to suggest the retention of
sulfinyl radical for that fragment ion, e.g. SO•bn+ or SO•yn .
+
Formation of Peptide Sulfinyl Radical Ions
Radical Attack of Inter-Chain Disulfide Bond
+
Studies have shown that molecular species (OH, N2, N2 , NO,
and NO2) as well as atomic species (metastable He, H, and O)
are abundant in an AP helium LTP [60, 61]. For peptides
having intra-chain disulfide bonds, one major product after the
nanoESI plume interacting with LTP were ions corresponding
to the addition of O and H to the intact peptide ([M + nH + OH]
n•+) [58, 59]. Note that since many oxidative radicals or species
coexisted in the helium LTP, the exact reaction mechanism for
this phenomenon was unclear. One possible route of forming
these products could come from the dissociative addition of
hydroxyl radical from LTP to the disulfide bond, resulting in
sulfinyl radical (−SO•) and sulfhydryl (−SH) at the cleavage
site. However, the location of the radical site from the above
system was ambiguous given that the sulfinyl radical could be
formed at either cysteine residue. In this study, peptides with
inter-chain disulfide bond were employed such that the two
peptide chains were separated after radical reactions and
peptide sulfinyl radical ions could be mass distinguished
(Scheme 1).
A typical reaction spectrum of the inter-chain disulfide
peptides in positive ionization mode is demonstrated with
peptide 1 (Figure 1a). Peptide 1 has two chains (A and B)
linked through a disulfide bond. The masses of the neutral A
and B chains (derived from a homolytic cleavage of the
disulfide bond) are indicated in the inset of Figure 1. Note that
before applying the plasma, only intact peptide ions could be
observed from nanoESI. When the LTP plasma and the
nanoESI were both functioning, the formation of sulfhydryl
(−SH) and sulfinyl radical (−SO•) from each peptide chain,
notated as [SHA + H]+, [SHB + H]+, [SO•A + H]+, and [SO•B + H]
+, could be clearly identified. Other minor reaction products
were also observed, such as −1 Da or −2 Da to the intact
peptide ions and separated chains, and consecutive oxidation
on amino acid side chains corresponding to n x 16 Da mass
increases [58, 59]. Compared with peptide radical cations,
Mass Spectrometry and AP Radical Reactions
Most experiments were performed on a 4000 QTRAP mass
spectrometer (Applied Biosystems, Toronto, Canada), with a
hybrid triple quadrupole/linear ion trap configuration. Two
types of collisional activation methods are available with this
instrument, beam-type CID and ion trap CID. For beam-type
CID, parent ions are isolated by Q1 quadrupole and accelerated
to Q2 for collisional activation. In ion trap CID, ion activation is
conducted in the Q3 linear ion trap with a dipolar excitation. In
this study, tandem mass spectrometric experiments were
facilitated by ion trap CID unless specified. Mass analysis was
performed in Q3 in linear ion trap mode. Typical parameters of
the mass spectrometry used were set as follows: spray voltage,
1400–1800 V; curtain gas, 10 psi; declustering potential, 20 V;
scan rate, 1000 Da/s. Data shown here were typical an average
of more than 50 scans. In order to conduct AP ion/radical
reactions, the nanoESI plume of peptide sample was allowed to
interact with the after-glow region of an AP helium low
temperature plasma (LTP) enabled in the side arm of a T-shaped
glass tube placed in front of the entrance of mass spectrometer
[58]. The schematics of the instrument and reaction setup are
shown in Scheme S-1 of the supplemental material. Accurate
mass measurements were performed on an LTQ-Orbitrap
(Thermo Fisher Scientific, San Jose, CA, USA) with a mass
resolution of 30,000 with an internal mass calibration.

L. Tan and Y. Xia: Peptide Sulfinyl Radical Ions
[SHA+H]+
+/-
589.3
Consecutive
oxidation
SO•A+H]+
ESI
[
621.3
radical rxns
(e.g. OH•)
604.3
+
+/-
(c)
(b)
Inter-chain
disulfide linked peptide
(a)
580
590
600
610
620
630
Site-Specific
Peptide Sulfinyl Radical Ion
m/z
Figure 2. Expanded view for chain A region derived from
nanoESI of Peptide 1 when the helium LTP source was
operated with increased helium flow rates: (a) 60 mL/min; (b)
90 mL/min; (c) 110 mL/min
Scheme 1. Formation of site-specific peptide sulfinyl radical
ion from radical attack of an inter-chain disulfide peptide. The
radical reactions allowed the formation of peptide sulfinyl
radical ions independent of ion polarity and charge state
rates on the formation of sulfinyl radical ions for the A chain
of peptide 1. Clearly, higher intensity of sulfinyl radical ions
were formed with a higher helium flow rate, presumably due
to higher number densities of radicals produced from more
intense plasma [60]. However, consecutive oxidation of
peptide side chain or reactions of the sulfinyl radical ions
became more competitive and the overall yield or purity of
the sulfinyl radical ions could be adversely affected [59], as
shown in Figure 2c. Therefore, it was preferred to keep a
moderate yield with moderate helium flow rate so that both
intensity and purity of sulfinyl radical could be guaranteed.
Under the optimized conditions, the relative intensity of
sulfinyl radical ions was about 30 % relative to the
remaining parent peptide ions.
fewer studies have been done on peptide radical anions
primarily due to limited ways of generating them in gas phase
[38–40, 42]. As shown in Figure 1b, peptide sulfinyl radical
anions could be generated simply by switching the ionization
mode from positive to negative since the reactions are induced
by radicals. The data shown in Figure 1 suggested that the
atmospheric ion/radical reaction approach allowed the flexibil-
ity of forming radical ions independent of ion charge polarity.
The yield of peptide sulfinyl radical ions could be
manipulated by changing the flow rate of helium in LTP.
The data in Figure 2 compare the effect of helium gas flow
[M+2H]2+
CLPTR A: 587.3 Da
524.5
100
(a)
HMAC
B: 459.2 Da
Radical Attack of Free Cysteine Residue
[SHA+H]+
589.3
50
0
An alternative way of forming peptide sulfinyl radical ions
is to directly oxidize peptides containing a single cysteine
residue from helium LTP. The reaction spectrum of
peptide 11 (sequence: CLPTR), equivalent to chain A of
peptide 1, is used as an example (Figure 3). The reactions
were optimized to give the best formation of sulfinyl
radical at m/z 604.3 in consideration of its intensity and
purity. The exact mechanism of forming the sulfinyl
radical from free thiol is not clear. Based on the radical
reactions of organic thiols, it can be a chain-reaction,in
which thiyl radical is formed first due to hydrogen
abstraction by hydroxyl radical and then quickly oxidized
to sulfinyl radical [53]. Molecular oxygen and NO2 were
reported to be involved in the oxidation process of thiyl
radicals [6]. The advantage of using free cysteine contain-
ing peptides for the formation of peptide sulfinyl radical
ions was that no additional procedure was needed to form
an inter-chain disulfide peptide. However, undesirable side
reactions giving rise to ions isobaric to the sulfinyl ions
were typically more competitive than situations using
inter-chain disulfide peptides. For instance, the m/z 604.3
[M+Na+H]2+
[M+K+H]2+
[SHB+H]+
[
SO•A+H]+
604.3
621.3
[
SO•B+H]+
461.3
637.3
476.3
450
500
550
600
650
m/z
[M-2H]2-
522.4
100
(b)
[
SHB-H]-
459.3
50
0
[
SHA-H]-
[
SO•B-H]-
587.5
[
SO•A-H]-
474.4
602.5
619.4
491.4
450
500
550
600
650
m/z
Figure 1. NanoESI mass spectra of peptide 1 when helium
LTP source was also operating: (a) positive ionization mode
and (b) negative ionization mode. The molecular weights of
neutral chain A and B due to homolytic cleavage of the
disulfide bond are shown in the inset of Figure 1a

L. Tan and Y. Xia: Peptide Sulfinyl Radical Ions
[
SO•A+H]+
Unimolecular Dissociation of Peptide Sulfinyl
Radical Cations
604.3
[M+H]+
589.4
100
CLPTR
605.3
603.3
Collisional activation was applied to a series of peptide
sulfinyl radical ions to gain insight into their gas-phase
unimolecular dissociation chemistry. Given the coexistence
of radical and charge, the competition of charge- versus
radical-directed fragmentation pathways is of special inter-
est. The ion trap CID spectra of four singly protonated
peptide sulfinyl radical ions are selected to represent the
general fragmentation behavior (Figure 4). In the first three
cases, each peptide contains one basic amino acid residue,
Arg, Lys, and His, respectively. Loss of 62 Da was the most
favorable channel. Accurate mass measurement of this loss
(61.9822 Da) corresponded to an elemental composition of
CH2SO (calculated exact mass: 61.9826 Da). This phenom-
enon was observed before in MS3 CID of [M + nH + OH]n•+
ions derived from intra-chain disulfide peptides [58]. The
62 Da loss was proposed to be radical driven, resulting from
a homolytic cleavage between the α- and β-carbons on the
sulfinyl radical side chain as shown in Scheme 2. Note that
this loss gives rise to glycyl radical at the original location of
cysteine, which is an important radical species in enzyme
chemistry [2]. Other channels such as losses of 17, 18, and
49 Da were also observed, corresponding to OH or NH3,
H2O, and HSO, respectively. A small degree of side chain
losses following the 62 Da loss were present as well. The
possible pathways for side chain loss ions have been
extensively studied for hydrogen deficient radical peptide
ions and therefore are not discussed in detail herein [26, 35,
62, 63]. The sulfinyl peptide radical ions were also subjected
621.3
50
0
[
SO•A+H]+
604.3
603.3
637.3
580
600
620
640
m/z
Figure 3. NanoESI mass spectrum of peptide 11(CLPTR)
when the helium LTP source was also operating. The inset
shows the zoomed view of sulfinyl ion region from Figure 1
(a), where peptide 1 was used to generate the same sulfinyl
radical ions
ions in Figure 3 contained a significant fraction of the 13C
isotope from m/z 603.3 ions, which was much less when
the inter-chain linked peptide was used (inset of Figure 3).
CID of m/z 603.3 showed a dominant 18 Da loss (data not
shown), which was very different from CID of the sulfinyl
radical ions at m/z 604.3. The identity of this species is
not clear at this moment. Under this kind of situation,
yield was compromised to ensure the formation of
relatively pure sulfinyl ions. In this study, peptides
containing both single inter-chain disulfide bonds and free
cysteines were employed to generate the peptide sulfinyl
radical ions.
- CH2SO
- CH2SO
(a)
100
(b)
100
317.2
542.3
[G(SO•C)GK + H]+
[(SO•C)LPTR+H]+
-62Da
379.2
-62Da
604.3
-56Da(L)
50
50
- HSO
-49Da
330.2
-18Da
586.3
486.2
0
0
200
300
400
400
500
600
+
- CH2SO
b2
(d)
(c)
215.1
414.2
[TL(SO•C) + H]+
100
50
0
100
50
0
[HMA(SO•C)+ H]+
-62Da
+
a2
351.2
476.2
-18Da
-74Da(M)
-47Da(M)
187.1
- CH2SO
-HSO
289.1
-18Da
427.1
+
b2
143.0
100
200
300
400
250
350
m/z
450
m/z
Figure 4. Ion trap CID of sulfinyl radical peptide ions: (a) [(SO•C)LPTR + H]+; (b) [G(SO•C)GK + H]+; (c) [HMA(SO•C) + H]+ ; and (d)
[TL(SO•C) + H]+

L. Tan and Y. Xia: Peptide Sulfinyl Radical Ions
-62(C)
(a)
100
50
0
CID
[(SO•C) G A I L R + H]+
+
647.4
-56(Ior L)
62 Da
loss
Sulfinyl radical
Glycyl radical
-43(L)
Scheme 2. Proposed pathway for the loss of 62 Da from CID
of peptide sulfinyl radical ions
100
300
500
700
+
y3
to beam-type CID and very similar fragmentation patterns to
that of ion trap CID were observed (data not shown).
However, the slightly different activation conditions (i.e.,
higher activation energies and shorter activation time for
beam-type CID, compared with ion trap CID, did bring
differences in the spectra. For example, higher intensities of
49 Da loss and sequential fragmentation were observed in
beam-type CID in many cases.
100
50
0
(b)
[(SO•C) G A I L R + 2H]2+
+
y2
324.2
-62(C)
+
so•b4
+
so•b3
+
y4
+
+
y1
y5
100
300
500
700
The fragmentation pathway for peptides not containing
any basic amino acid residue is quite different from the
peptide sulfinyl radical cations containing basic amino acid
residue(s). The MS2 ion trap CID data of [TL(SO•C) + H] + is
shown as an example in Figure 4d. The 62 Da loss turns out
to be very minor, whereas charge directed peptide backbone
fragmentation (forming b2 and a2 ions) is more abundant in
the spectrum. It is unclear at this point if the activation
barrier for the radical-directed 62 Da loss is affected by
charge or not. However, the amide bond cleavages are
facilitated by protons as being clearly depicted by the
“mobile proton” theory [64, 65]. Basic amino acid residues
(R, K, and H), which sequester protons in various degrees,
are known to elevate activation energies for peptide amide
bond cleavages [64, 66]. It is understandable, therefore, that
the amide bond cleavages are less competitive to the radical-
directed loss in peptide sulfinyl radical ions having basic
amino acid residues (R, K, and H). This trend was
consistently observed for over 20 singly protonated peptide
sulfinyl radical ions. Similar phenomenon was observed for
CID of arginine containing tripeptide radical cations [66–
68]. Theoretical calculations by Zhao et al. showed that the
energy barriers for radical migration were lowered by 5–
10 kcal/mol due to localization of charge on arginine side-
chain, while the charge-directed dissociation barriers were
increased significantly [67]. The major CID products of
singly protonated sulfinyl radical peptides with at least one
basic amino acid residue are summarized in Tables S-1
(supplemental material). Table S-2 summarizes CID of
singly protonated species without basic amino acid residue.
m/z
Figure 5. MS2 ion trap CID of (a) singly protonated (m/z
647.4) and (b) doubly protonated (m/z 324.2) peptide sulfinyl
radical cations having a sequence of (SO•C)GAILR
data in Figure 5 shows an example of CID of singly versus
doubly protonated peptide sulfinyl radical ions having a
sequence of (SO•C)GAILR. The extent of parent ion
dissociation was kept at similar degree for comparison.
Clearly, the fragmentation patterns were drastically different
for 1+ and 2+ charge states. For the singly charged species
(Figure 5a), radical-directed 62 Da loss was the most
abundant fragment leading to the formation of glycyl radical
-62
100
(a)
[(SO•C) S R + H]
+
50
380.2
-17
0
200
250
300
350
400
-49
100
(b)
[(SO•C) S R - H]-
50
0
378.2
-62
-17
Charge State Effect
The method of utilizing AP radical reactions also allowed
facile formation of peptide sulfinyl radical ions with
different charge states given that the multiple charge states
could be observed for the intact peptide ions. This capability
enhanced further investigation of the effect of ion charge
states on radical- versus charge-directed fragmentation. The
200
250
300
m/z
350
400
Figure 6. MS2 ion trap CID of (a) singly protonated (m/z
380.2) and (b) singly deprotonated (m/z 378.2) peptide sulfinyl
radical ions with a same sequence of (SO•C)SR

L. Tan and Y. Xia: Peptide Sulfinyl Radical Ions
ions. Sequential side-chain losses from the thus formed glycyl
radical ions, such as loss of 43 Da from leucine and loss of
56 Da from either leucine or isoleucine [62, 63], were observed
as minor fragmentation channels. In the CID of doubly
protonated ions (Figure 5b), the loss of 62 Da was largely
suppressed, while abundant b and y ions were observed. The
distinct fragmentation behavior suggested that charge state
(number of mobile protons) did affect the competition for
radical versus charge directed fragmentation pathways. Similar
to the argument made for basic amino acid residue effect, the
availability of mobile protons in higher charge states effective-
ly lowered the activation barrier for amide bond cleavages and
resulted in forming abundant b and y ions. The observation of
stabilization from the captodative effect [27, 70]. On the
other hand, the formation of dehydroalanine from 49 Da loss
gives rise to a stable even-electron structure for anions. The
main fragmentation channels for another five sulfinyl radical
anions are summarized in Table S-4. The suppressed loss of
62 Da was consistently observed. In some cases loss of
93 Da was also observed. MS3 CID of the 49 Da loss
showed a dominant 44 Da loss (Figure S-1, supplemental
material), suggesting that the 93 Da loss was mainly due to
sequential fragmentation.
Conclusions
+
yn (n=1–5), SO•b3 , and SO•b4+ (superscript “SO•” indicates the
Radical attack to either an inter-chain disulfide bond or a
free cysteine thiol group within a peptide was enabled inside
an AP reactor in front of the inlet of a mass spectrometer.
The AP radical reactions allowed a facile means of forming
a new type of site-specific peptide radical ion species,
sulfinyl radical ions, in both positive and negative ion modes
and in various ion charge states. Low energy collisional
activation of peptide sulfinyl radical cations revealed that
proton mobility strongly affected the competition of radical-
directed side-chain losses against charge-directed backbone
fragmentation. For systems with reduced proton mobility
(singly protonated ions containing basic amino acid resi-
dues), radical initiated 62 Da loss (CH2SO) dominated,
likely due to the elevated activation barrier for proton
facilitated backbone fragmentation. However, for systems
with mobile protons, b and y ions from charge directed
backbone fragmentation were prevalent. Loss of 62 Da led
to the formation of glycyl radical at the initial cysteine
residue, which would be convenient in generating hydrogen
deficient peptide radical species with known initial radical
site location. Charge polarity was also found to play an
important role in the dissociation behavior of peptide
sulfinyl radical ions. In negative ion mode, a major loss of
49 Da (HSO) instead of 62 Da was observed, which might
be due to the stability issue of final product. Future studies
will focus on reactivity of sulfinyl radicals toward com-
pounds containing unsaturated functional groups, disulfide
bond, and sulfhydryl group, which are known to be
vulnerable under radical attack.
retention of sulfinyl radical on the fragments) in Figure 5b
demonstrated that the sulfinyl radical was retained on the
original cysteine side-chain within the backbone fragments.
The activation energy for peptide amide bond cleavage was
reported to be within 25 to 40 kcal/mol range [69]. Therefore, it
could be inferred that the activation energy of the radical
directed 62 Da loss was in comparable range to that of amide
bond cleavage. Small sulfinyl radical ion systems are currently
under investigation to provide a clearer picture on sulfinyl
radical ion structures and energetics.
More examples of charge state effect are summarized in
Table S-3. A consistent trend of suppressed 62 Da loss and
increased backbone fragmentation was observed for higher
charge states where mobile protons were available. Hess et al.
studied the fragmentation of singly, doubly, and triply charged
hydrogen deficient peptide radical cations formed via infrared
multiphoton dissociation (IRMPD) and electron induced
dissociation (EID). They also indicated that backbone frag-
mentations were highly dependent on the charge states [37].
Charge Polarity Effect
The charge polarity was found to play an important role in
the unimolecular fragmentation of sulfinyl radical ions. A
comparison of collisional activation of peptide sulfinyl
radical cations and anions sharing the same sequence
(CSR) is shown in Figure 6. For the singly protonated
species (Figure 6a), loss of 62 Da was dominant as expected.
However, the spectrum changed dramatically by only
switching from positive mode to negative mode. The
sulfinyl radical anion gave very little loss of 62 Da but
abundant loss of 49 Da. Accurate measurement confirmed
that it was the loss of HSO. One possible pathway
accounting for the loss of HSO might be due to a C–S
cleavage accompanied by an α-carbon hydrogen abstraction
leading to the formation of dehydroalanine at the initial
cysteine site. The different behavior of the protonated versus
deprotonated peptide radical ions might be explained by the
stability of product ions formed in different charge polarities.
The loss of 62 Da leads to the formation of a glycyl radical.
It has been suggested that peptide glycyl radical anion is less
stable compared to glycyl radical cation due to the lack of
Acknowledgments
The authors acknowledge support for this research Purdue
Research Fund. The authors also thank Professor R. G.
Cooks for the use of LTQ Orbitrap for accurate mass
measurements.
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