Protein Carbonylation Detected with Light and Heavy Isotope-Labeled 2,4-Dinitrophenylhydrazine by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
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概要
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Oxidation of proteins leads to carbonylation―the formation of aldehydes or ketones―at the amino acid side chain and/or the terminal amino groups. Carbonylated proteins have been conventionally detected by UV absorption spectrometry of the stable adduct with 2,4-dinitrophenylhydrazine (DNPH). However, this routine method is limited to detection of the total carbonyl content and does not provide structural information. We developed an isotope-dilution method for the specific detection of carbonylated proteins using ^<12>C_6-DNPH and ^<13>C_6-DNPH. This method has the following steps: the oxidized protein or peptide is divided into two parts, and these parts are independently labeled with ^<12>C_6-DNPH and ^<13>C_6-DNPH; the mixtures of these two labeled solutions are subsequently measured with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The carbonylated peptide was found by searching for a doublet peak having a mass difference of 6 Da. We examined oxidized angiotensin I and oxidized lysozyme prepared by treatment with NaOCl. The oxidized angiotensin I showed four pairs of doublet peaks in the MALDI-TOF mass spectrum. The structure was determined by tandem mass spectrometry. In the case of tryptic digest of the oxidized lysozyme, two carbonylated products could be easily identified even in a complex mixture. The use of ^<13>C_6-DNPH provides rapid and accurate detection of carbonylated peptides even in complex mixtures.
- 日本質量分析学会の論文
- 2009-12-01
著者
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OSAKA Issey
Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology
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Osaka Issey
Center For Nano Materials And Technology Japan Advanced Institute Of Science And Technology (jaist)
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Osaka Issey
Center For Nano Materials And Technol. Japan Advanced Inst. Of Sci. And Technol.
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TSUJIMOTO Kazuo
School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST)
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Matsumoto Hiroyuki
Department of Electrical Engineering, Toyo University
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Tsujimoto Kazuo
School Of Material Sciences Japan Advanced Institute Of Science And Technology
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KINUMI Tomoya
Bio-medical Standard Section, National Metrology Institute of Japan, National Institute of Advanced
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HAYASHI Akio
Agilent Technologies
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KAWAI Takaaki
School of Material Sciences, Japan Advanced Institute of Science and Technology
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Tsujimoto Kazuo
School Of Material Sciences Japan Advanced Inst. Of Sci. And Technol.
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Kawai Takaaki
School Of Material Sciences Japan Advanced Institute Of Science And Technology
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Kinumi Tomoya
Bio-medical Standard Section National Metrology Institute Of Japan National Institute Of Advanced In
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Kinumi Tomoya
Bio-medical Standard Section National Metrology Institute Of Japan (nmij) National Institute Of Adva
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Matsumoto Hiroyuki
Department Of Biochemistry And Molecular Biology University Of Oklahoma Health Sciences Center
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Kinumi Tomoya
Bio-medical Standard Section National Metrology Inst. Of Japan National Inst. Of Advanced Industrial
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Matsumoto Hiroyuki
Department Of Biochemistry And Molecular Biology The University Of Oklahoma Health Sciences Center
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