Mass Spectroscopic Characterization of Protein Modification by Malondialdehyde

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• 作者：Takeshi Ishii ; Shigenori Kumazawa ; Toyo Sakurai ; Tsutomu Nakayama ; and Koji Uchida
• 刊名：Chemical Research in Toxicology
• 出版年：2006
• 出版时间：January 2006
• 年：2006
• 卷：19
• 期：1
• 页码：122 - 129
• 全文大小：155K
• 年卷期：v.19,no.1(January 2006)
• ISSN：1520-5010

文摘

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane by lipidperoxidation, is a strong alkylating agent of primary amino groups. We recently raised a monoclonalantibody (mAb1F83) directed to the lipofuscin-like MDA-lysine adduct and demonstrated the presenceof immunoreactivity to the antibody in the atherosclerotic lesions, in which intense positivity was associatedprimarily with macrophage-derived foam cells (Yamada et al., (2001) J. Lipid Res. 42, 1187-1196). Toidentify the structure of the epitope in the protein recognized by mAb1F83, in the present study, weexposed chain B from bovine insulin (insulin B chain) to MDA and characterized the MDA adducts bymass spectrometry. The MDA-modified insulin B chain was digested with V8 protease, and the resultingpeptides were subjected to liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). The MS/MS analyses confirmed the formation of N-propenal- (+54 Da) and dihydropyridine-type (DHP, +134 Da) adducts in both Lys²⁹ and the N-terminus of insulin B chain. The ELISAanalysis of HPLC fractions of peptides, including the DHP adducts using mAb1F83, showed that theimmunoreactivity of the DHP-lysine adduct was more significant than the DHP-N-terminus adduct.The results of this study chemically characterized that the MDA adducts such as DHP-type adductsgenerated in the -amino group of lysine and N-terminal amino acid residues in the protein and thestructure of the epitope recognized by mAb1F83 were DHP-lysine adducts in protein.