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Please use this identifier to cite or link to this item: http://hdl.handle.net/10119/8498

Title: Protein Carbonylation Detected with Light and Heavy Isotope-Labeled 2,4-Dinitrophenylhydrazine by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Authors: KINUMI, Tomoya
OSAKA, Issey
HAYASHI, Akio
KAWAI, Takaaki
MATSUMOTO, Hiroyuki
TSUJIMOTO, Kazuo
Keywords: Oxidation
Dinitrophenylhydrazine
Stable isotope-labeling
MALDI
Angiotensin
Issue Date: 2009
Publisher: 日本質量分析学会
Magazine name: Journal of the Mass Spectrometry Society of Japan
Volume: 57
Number: 6
Start page: 371
End page: 377
Abstract: 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.
Rights: Tomoya KINUMI, Issey OSAKA, Akio HAYASHI, Takaaki KAWAI, Hiroyuki MATSUMOTO, and Kazuo TSUJIMOTO (2009) Protein Carbonylation Detected with Light and Heavy Isotope-Labeled 2,4-Dinitrophenylhydrazine by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry, Journal of the Mass Spectrometry Society of Japan, 57(6), 371-377. ここに掲載した著作物の利用に関する注意:本著作物の著作権は日本質量分析学会に帰属します。本著作物は著作権者である日本質量分析学会の許可のもとに掲載するものです。
URI: http://hdl.handle.net/10119/8498
Material Type: publisher
Appears in Collections:i10-1. 雑誌掲載論文 (Journal Articles)

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