Research Article
Received: 15 November 2009
Revised: 9 December 2009
Accepted: 22 December 2009
Published online in Wiley Interscience:
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
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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
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J. Pept. Sci. 2010; 16: 148–152
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.