Determination of protein-bound methionine oxidation in the hippocampus of adult and old rats by LC-ESI-ITMS method after microwave-assisted proteolysis |
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Authors: | Long Li-Hong Wu Peng-Fei Guan Xin-Lei Zhang Jun-Qi Jin You Zhang Zui Wang Yue Li Yi-Yong Chen Jian-Guo Wang Fang |
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Institution: | (1) Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China;(2) The Key Laboratory of Neurological Diseases of the Ministry of Education of China, Huazhong University of Science and Technology, Wuhan, 430030, China;(3) The Key Laboratory of Natural Drug Chemistry and Evaluation of Hubei Province, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China |
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Abstract: | Protein-bound methionine (Met) oxidation has been associated with normal aging and a variety of age-related diseases, including
Alzheimer’s disease and Parkinson’s disease. Monitoring the changes of protein-bound methionine content in the brain in response
to normal aging and oxidative stress is of great interest and could be used as an indicator of oxidative stress of rats in
pathological conditions. We have developed a rapid analytical method for the determination of oxidized products of protein-bound
methionine in rat brain. The assay involved rapid acid proteolysis with microwave irradiation and solid-phase extraction of
the free amino acids followed by LC-ESI-ITMS analysis. Detection was achieved in positive ionization with an ion trap mass
spectrometer operating in multiple-reaction monitoring mode. The calibration curves of the analytes were linear (r
2 > 0.99) in the range between 0.098 and 1.560 μg/mL. Intra- and inter-day relative standard deviation percentages were <9%
and <8%, respectively. The assay performance was sufficient to support a rapid analytical tool for monitoring brain protein-bound
methionine oxidation levels. The content of protein-bound Met and methionine sulfoxide (MetO) in the hippocampus of adult
and old rats with or without H2O2 treatment was determined by employing the new method. The content of protein-bound MetO was significantly increased in old
rats after exposure to H2O2. This result indicates increased sensitivity to Met oxidation in the hippocampus of old rats. |
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