The oxidative products of methionine as site and content biomarkers for peptide oxidation

Article abstract of DOI:10.1002/psc.1212

Biomarkers for peptide/protein oxidation under oxidative stress (OS) hold both incredible application potential as well as significant challenges. In this article, liquid chromatography and mass spectrometry were applied to establish a new method for evaluating the oxidation site and degree of peptide oxidized, with its oxidative product serving as biomarker. In the three model peptides, peptide FMRF (containing a methionine) was prone to undergo oxygen addition under UV/H₂O₂ oxidization, forming a sulfoxide (FM(O)RF) with a stable chromatographic peak separate from the model peptides. The oxidation content of FMRF, expressed as S_FM(O)RF/(S_FM(O)RF + S_FMRF), is positively correlated with oxidation time. Based on sequence analysis of FM(O)RF, the oxidation mechanism (site and extent) of FMRF under UV/H₂O₂ oxidization was explicitly clarified. By comparing the specific injury to eachmodel peptide, we found that the oxidative products of Met-containing peptides are good biomarkers for OS. This research not only expands the range of biomarkers for OS, but also provides an efficient and accurate method for evaluating oxidation damage to peptides and even proteins. Copyright

Full text of DOI:10.1002/psc.1212

Research Article
Received: 15 November 2009
Revised: 9 December 2009
Accepted: 22 December 2009
Published online in Wiley Interscience:
(www.interscience.com) DOI 10.1002/psc.1212
The oxidative products of methionine as site
and content biomarkers for peptide oxidation
Wansong Zong, Rutao Liu,∗ Meijie Wang, Pengjun Zhang, Feng Sun
and Yanmin Tian
Biomarkers for peptide/protein oxidation under oxidative stress (OS) hold both incredible application potential as well as
significant challenges. In this article, liquid chromatography and mass spectrometry were applied to establish a new method
for evaluating the oxidation site and degree of peptide oxidized, with its oxidative product serving as biomarker. In the three
model peptides, peptide FMRF (containing a methionine) was prone to undergo oxygen addition under UV/H2O2 oxidization,
forming a sulfoxide (FM(O)RF) with a stable chromatographic peak separate from the model peptides. The oxidation content
of FMRF, expressed as SFM(O)RF/(SFM(O)RF + SFMRF), is positively correlated with oxidation time. Based on sequence analysis of
FM(O)RF, the oxidation mechanism (site and extent) of FMRF under UV/H2O2 oxidization was explicitly clarified. By comparing
the specific injury to each model peptide, we found that the oxidative products of Met-containing peptides are good biomarkers
for OS. This research not only expands the range of biomarkers for OS, but also provides an efficient and accurate method for
c
evaluating oxidation damage to peptides and even proteins. Copyright ꢀ 2010 European Peptide Society and John Wiley &
Sons, Ltd.
Keywords: oxidative stress; biomarker; peptides; UV/H2O2 oxidation; LC–MSn; oxidative product
Introduction
dependent [15–18]. However, these products have not been
treated as potential biomarkers for OS. In this study, liquid
chromatography/mass spectrometry (LC/MS) and MS/MS assays
were developed to examine the feasibility of using peptide
oxidation products as biomarkers to probe the oxidation sites
and oxidation degrees of peptides. The oxidation of target
peptides was simulated by exposing peptides to UV/H2O2
oxidation, then the peptides and their oxidative products were
separated and identified by LC/MS. The sites and degree
of oxidation were further obtained by LC/MS and MS/MS
analyses.
Under endogenous or exogenous stimulation, excessively pro-
duced reactive oxygen species (ROS), such as H2O2, HO, HO2,
HClO, and O3 can destroy the dynamic equilibrium between ox-
idants and anti-oxidant systems and induce oxidative stress (OS)
[1–5]. In the case of OS, ROS can oxidize biomolecules (lipids,
nucleic acids, peptides, and proteins) and thus induce apop-
tosis, cancer, arteriosclerosis, and other diseases [2,4–6]. Thus,
clarifying oxidation mechanisms of biomolecules caused by OS
not only favors understanding of disease processes, but also is
beneficial to disease prevention, early diagnosis and treatment
[4,6,7].
Biomarkers can reflect the physiological, biochemical, immuno-
logical, and genetic characteristics in the oxidation processes,
and therefore they are key indexes for evaluating oxidative dam-
age induced by OS [7–14]. Compared with the widely studied
biomarkers for lipid [9,11] or nucleic acid oxidation [13], biomark-
ers for peptide and protein oxidation has been a limiting factor
in research on the oxidation mechanisms [8,9]. Although 2,4-
dinitrophenylhydrazine, nitrotyrosine, and dityrosine have been
widely used as biomarkers for peptide and protein oxidation,
the traditional spectroscopic and immunological techniques for
detecting them have low sensitivity and can only determine the
total carbonyls, nitrotyrosine, or dityrosine in oxidized peptides
[9,11]. In addition, they cannot provide specific information for
the oxidation sites, let alone the mechanism and degree of oxi-
dation for each site. As such, novel biomarkers that can indicate
the oxidation site and oxidation degree for peptides have un-
paralleled advantages in clarifying the oxidation mechanisms of
peptides.
Materials and Methods
Materials
Peptides Phe-Met-Arg-Phe (FMRF, 598.76 Da), Asp-Arg-Val-Tyr-
Val-His-Pro-Phe (DRVYVHPF, 1032.18 Da), and Arg-Pro-Pro-Gly-
Phe-Ser-Pro-Tyr-Arg (RPPGFSPYR, 1076.23 Da) were purchased
from GL Biochem Inc. (Shanghai, China) and had a purity at least
of 95%. Thiourea, 30% H2O2, methionine (Met), and trifluoroacetic
acid (TFA) were ordered from Sinopharm Chemical Reagent Inc.
(Shanghai, China). HPLC methanol and HPLC acetonitrile were
purchased from Merck (Germany). All reagents were prepared
with Millipore ultrapure water and no buffer was used.
∗
Correspondence to: Rutao Liu, School of Environmental Science and Engi-
neering, Shandong University, Jinan 250100, People’s Republic of China.
E-mail: rutaoliu@sdu.edu.cn
Tandem mass spectrometry (MS/MS) research has confirmed
that peptides can be oxidized, forming carbonyl, hydroxyl,
and sulfoxide containing products that are stable and time-
School of Environmental Science and Engineering, Shandong University, 27
Shanda South Road, Jinan 250100, People’s Republic of China
c
J. Pept. Sci. 2010; 16: 148–152
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

NEW BIOMARKERS FOR PEPTIDE OXIDATION
mobile phase B was gradually reduced to 50% over 15 min. After
a 5-min isocratic elution, mobile phases were rapidly switched to
the initial conditions to equilibrate the column for 10 min. The
column temperature was set at 30 ◦C.
LC/MS was performed on a micromass ZQ mass spectrometer
(Waters) operated with the electrospray ionization (ESI) source in
positive ion mode. The ESI source conditions were set as follows:
capillary voltage 3.5 kV, sample cone voltage 55 V, extraction cone
voltage 0.5 V, source temperature 110 ◦C, and cone gas (N2) 30 l/h.
The data were processed using MassLynx software, version 4.1.
MS/MS Analysis of Target Peptides
MS/MS were also obtained in positive ion mode by direct injection
of samples into an LCQ Fleet mass spectrometer (ThermoFisher,
USA) using a syringe pump at 5 µl/min. During the MS/MS scan,
the collision energy was set at 40 eV and the MS/MS scan range
was automatically adjusted according to the molecular weight of
the parent ion.
Figure 1. LC/MS chromatograms of (A) the control sample and (B) the
oxidized sample exposed to UV/H2O2 oxidation for 15 min. Peak a,
RPPGFSPYR; b, DRVYVHPF; c, FMRF; and d, FMRF + 16 Da.
Results and Discussion
LC/MS Analysis of the Native and Oxidized Peptides –
Discovery of the Oxidative Products from Model Peptides
UV/H2O2 Oxidation of Model Peptides
Immediately after mixing 200 µL (60 pmol/µL) DRVYVHPF, RPPGF-
SPYR and FMRF with 20 µL 1.5% H2O2, the samples were exposed
to a 100 W UV lamp. After exposure to radiation for a certain
period of time, the oxidation reaction was stopped by adding
340 µl stop solution (thiourea 100 mmol/l and Met 2 mmol/l). Stop
solution was also added in to the control sample to prevent the
post-oxidation in the processes of sample preparation and chro-
matographic separation. All samples were stored at 4 ◦C before
LC/MS or MS/MS analysis.
The chromatographic separation of model peptides and their
oxidative products, coupled with the full scan mass spectrum of
corresponding chromatography peaks, led to the identification of
oxidative products of the model peptides.
It can be seen from Figure 1 (curve A) that the intact model
peptides have three, well-separated characteristic peaks between
19 and 25 min, with out evident oxidation. The full scan mass
spectra of these successively eluted peaks (Figure 2) verify
that peaks a, b, and c correspond to peptides DRVYVHPF,
RPPGFSPYR, and FMRF, respectively. For samples exposed to
UV/H2O2 oxidation,anewpeakappearedat20.84 min,adecreased
intensity peak was observed for FMRF, while DRVYVHPF and
RPPGFSPYR remained unchanged (curve B, for 5 min oxidation).
The new peak must be associated with the oxidation of FMRF.
Comparing their MS results, peak d (m/z of 616 Da) has an increase
of 16 Da over FMRF (m/z of 600 Da), indicating a difference in
oxygen atom compared with that of FMRF.
LC/MS Analysis of Native and Oxidized Peptides
Chromatographic separations were performed on a HPLC (Waters
2695) and the mobile phases used were: (i) 90% acetonitrile and
(ii) 10%acetonitrile.Bothmobilephasescontain0.1%TFA.Samples
(10 µl) were loaded onto a Great Eur-Asia C18 column (4.6 mm ×
250 mm, 5 µm particle size, 120 Å) then eluted at a flow rate of
0.5 ml/min. Peptides were eluted using 100% B for 6 min, then
Figure 2. Full scan mass spectra of the corresponding chromatographic peaks in Figure 1 (see Figure 1 for assignments). This figure is available in colour
online at www.interscience.wiley.com/journal/jpepsci.
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J. Pept. Sci. 2010; 16: 148–152 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ZONG ET AL.
We also found that the peptides DRVYVHPF and RPPGFSPYR
could be oxidized by UV/H2O2, forming oxidation products with
an added O atom. However, these oxidation products have lower
abundance and are not separated from total ion chromatogram
(TIC) peaks of the model peptides. The Met residue is more
reactive to UV/H2O2 oxidation than the other amino acid residues
in the model peptides [3,5]. A valid biomarker should appear in
a sufficient amount to be easily detected and to have a highly
efficient separation. Therefore, the oxidation product (with an
added O atom) of FMRF was selected as a prospective biomarker
for OS.
105
100
95
A
25
20
15
10
5
MS/MS Analysis of the Native and Oxidized Peptides –
Identification of the Oxidation Site
0
200
300
400
m/z
500
600
Tandem mass spectrometry (MS/MS) is widely used to analyze
peptide sequences by comparing the molecular weights of a set
of interdependent fragment ions. This superior strategy can also
be used to identify the oxidation sites of peptides. Comparing the
MS/MS spectra of FMRF and its oxidation product (Figure 3), we
find: a shift of +16 Da is detected for the b2, b3, and y3 fragment
ions, suggesting the oxidation site is located at the Met residue.
The S atom in the Met residue is the main target site of OS (much
more active than other atoms), and is always oxidized into a
sulfoxide (–CH2SCH3 is oxidized into –CH2SOCH3) [3,19]. So FMRF
is likely to be oxidized into FM(O)RF.
UnderUV/H2O2 oxidation,H2O2,UV,andHO/HO2·(decomposed
fromH2O2)canserveasthemainoxidizingagentsfortheoxidation
of FMRF [20,21]. Therefore, evaluating their contributions to
FM(O)RF will clarify the oxidation mechanism of FMRF. When
a mixture of the three model peptides is oxidized by H2O2 or
UV alone, the corresponding TICs (Figure 4) also have a new
peak near 20.84 min for FM(O)RF and a decreased peak for FMRF.
Thus, both H2O2 and UV can oxidize FMRF. By comparing the
oxidative content of FMRF produced by UV, H2O2, and UV/H2O2,
the contribution of HO/HO2·(decomposed from H2O2) can be
identified. In the samples oxidized by UV, H2O2, and UV/H2O2 for
20 min, the degree of oxidation of FMRF by UV or H2O2 alone
(3.7 and 16.4%) is much less than the 37.5% oxidation induced
by the combination of UV/H2O2 (shown in Figure 5). The total
amount oxidized by H2O2 and UV individually (20.1%) is also
less than 37.5%. The difference (17.4%) should be attributed to
the HO/HO2·formed by irradiating H2O2 with UV light. Thus the
oxidation of FMRF under UV/H2O2 oxidation can be achieved in a
variety of ways (described in Scheme 1).
B
100
15
10
5
0
200
300
400
m/z
500
600
Figure 3. (A) MS/MS spectrum of the protonated FMRF at m/z 600.29
0.5 Da. (B) MS/MS spectrum of the protonated FMRF
16O at
m/z 616.29 0.5 Da. This figure is available in colour online at
www.interscience.wiley.com/journal/jpepsci.
+
Quantitation of the Oxidized Peptide – Validation of the
Proposed Biomarker Candidate
In LC/MS analysis, the relative amounts of analytes (peptides),
usually expressed by their TIC peak areas, depend on their
protonation efficiency [22,23]. The arginine residue in FMRF or
FM(O)RF is the main site where the proton is located, but is not
the main oxidative site. So UV/H2O2 oxidation has no significant
Figure 4. (A) LC/MS chromatogram of the oxidized sample exposed to H2O2 for 20 min. (B) LC/MS chromatogram of the oxidized sample exposed to UV
light for 20 min. (C) Averaged full scan mass spectrum corresponding to peak ‘d’ in (A). (D) Averaged full scan mass spectrum corresponding to peak ‘d’
in Figure 1(B). This figure is available in colour online at www.interscience.wiley.com/journal/jpepsci.
c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 148–152

NEW BIOMARKERS FOR PEPTIDE OXIDATION
50
40
32
24
16
8
37.5%
40
30
20.1%
16.4%
20
10
0
0
0
5
10
15
20
25
Oxidation time (min)
3.7%
UV
Figure 6. Dose–response curve for the oxidation degree of FMRF exposed
to UV/H2O2 oxidation with the extended time.
H2O2
UV+H2O2
UV/H2O2
a sulfoxide containing peptide FM(O)RF. The oxidized content of
FMRF, expressed as SFM(O)RF/(SFM(O)RF +SFMRF), is linearly correlated
with oxidation time for the first 15 min. The rapid separation and
accurate identification of FM(O)RF indicate that oxidized peptides
can be treated as novel biomarkers to evaluate the sites and
degrees of oxidation of peptides. By comparing the specific state
of each model peptide, we also found that oxidative products
of Met-containing peptides could be a suitable biomarker for
OS. Coupling with enzymatic digestion, this technique can be
further used to screen biomarkers for evaluating the oxidative
mechanisms of proteins.
Figure 5. Oxidation degrees of FMRF induced by H2O2, UV and UV/H2O2
for 15 min.
Acknowledgements
ThisworkissupportedbyNSFC(20607011,20875055),theFokYing
Tong Education Foundation (111082), NCET-06-0582 and Foun-
dation for Middle Young Scientists, and Key Science-Technology
Project in Shandong Province (2007BS08005, 2008GG10006012)is
also acknowledged. The authors thank Dr Pamela Holt for editing
the manuscript for English.
Scheme 1. Oxidation mechanisms of FMRF induced by UV/H2O2.
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