Mass spectroscopic characterization of protein modification by malondialdehyde
Mass spectroscopic characterization of protein modification by malondialdehyde
复制标题
DOI：
10.1021/tx050231p
复制
发表时间：
2006-01-01
影响因子：
4.1
通讯作者：
Uchida, K
中科院分区：
文献类型：
--
作者：
Ishii, T;Kumazawa, S;Uchida, K
丙二醛 (MDA) 是一种通过脂质过氧化在膜中产生的天然二醛，是伯氨基的强烷化剂。我们最近提出了一种针对脂褐素样 MDA-赖氨酸加合物的单克隆抗体 (mAb1F83)，并证明在动脉粥样硬化病变中存在对该抗体的免疫反应性，其中强烈的阳性反应主要与巨噬细胞衍生的泡沫细胞相关 (Yamada 等人，(2001) J. Lipid Res. 42, 1187-1196)。为了鉴定mAblF83识别的蛋白质中表位的结构，在本研究中，我们将牛胰岛素的B链（胰岛素B链）暴露于MDA，并通过质谱法表征MDA加合物。 MDA 修饰的胰岛素 B 链用 V8 蛋白酶消化，所得肽进行液相色谱-电喷雾电离-质谱 (LC-ESI-MS/MS)。 MS/MS 分析证实在 Lys(29) 和胰岛素 B 链的 N 末端形成了 N-丙烯醛（+54 Da）和二氢吡啶型（DHP，+134 Da）加合物。使用 mAb1F83 对肽（包括 DHP 加合物）的 HPLC 级分进行 ELISA 分析，结果表明 DHP-赖氨酸加合物的免疫反应性比 DHP-N 末端加合物更显着。本研究结果从化学上表征，蛋白质中赖氨酸ε氨基和N端氨基酸残基生成的DHP型加合物等MDA加合物以及mAb1F83识别的表位结构均为蛋白质中的DHP-赖氨酸加合物。
Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane by lipid peroxidation, is a strong alkylating agent of primary amino groups. We recently raised a monoclonal antibody (mAb1F83) directed to the lipofuscin-like MDA-lysine adduct and demonstrated the presence of immunoreactivity to the antibody in the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells (Yamada et al., (2001) J. Lipid Res. 42, 1187-1196). To identify the structure of the epitope in the protein recognized by mAblF83, in the present study, we exposed chain B from bovine insulin (insulin B chain) to MDA and characterized the MDA adducts by mass spectrometry. The MDA-modified insulin B chain was digested with V8 protease, and the resulting peptides 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 Lys(29) and the N-terminus of insulin B chain. The ELISA analysis of HPLC fractions of peptides, including the DHP adducts using mAb1F83, showed that the immunoreactivity 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 adducts generated in the epsilon-amino group of lysine and N-terminal amino acid residues in the protein and the structure of the epitope recognized by mAb1F83 were DHP-lysine adducts in protein.