148 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/H 2 O 2 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/H 2 O 2 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 evaluating oxidation damage to peptides and even proteins. Copyright c  2010 European Peptide Society and John Wiley & Sons, Ltd. Keywords: oxidative stress; biomarker; peptides; UV/H 2 O 2 oxidation; LC – MSn; oxidative product Introduction Under endogenous or exogenous stimulation, excessively pro- duced reactive oxygen species (ROS), such as H 2 O 2 , HO, HO 2 , HClO, and O 3 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. 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- 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/H 2 O 2 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. 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% H 2 O 2 , 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 School of Environmental Science and Engineering, Shandong University, 27 Shanda South Road, Jinan 250100, People’s Republic of China J. Pept. Sci. 2010; 16: 148–152 Copyright c  2010 European Peptide Society and John Wiley & Sons, Ltd.