Screening Assay for Metal-Catalyzed Oxidation Inhibitors Using Liquid Chromatography-Mass Spectrometry with an N-Terminal β-Amyloid Peptide
Screening Assay for Metal-Catalyzed Oxidation Inhibitors Using Liquid Chromatography-Mass Spectrometry with an N-Terminal β-Amyloid Peptide
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DOI：
10.1021/ac802162n
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发表时间：
2009-03-01
影响因子：
7.4
通讯作者：
Oka, Hisao
中科院分区：
文献类型：
--
作者：
Inouo, Koichi;Nakagawa, Ako;Oka, Hisao
金属结合淀粉样蛋白β（A β）肽产生微区域催化活性氧（ROS）介导阿尔茨海默病的神经毒性，这种活性的抑制剂可能具有治疗价值。目前没有针对金属结合肽产生的特异性ROS抑制剂的分析方法。我们报告了一种基于液相色谱-质谱（LC-MS）的金属催化氧化（MCO）抑制剂的筛选方法，该方法使用模型N-末端A β肽（A β（1-6））。当通过Cu（II）/抗坏血酸进行MCO时，分别在m/z 729.2和364.8以及m/z 685.3和343.3处观察到单电荷和双电荷A β（1-6）分子，与模型肽相比，对应于质量减少45和89 Da。相反，H2 O2不修饰A β（1-6）肽。修饰肽的特征在于A β（1-6）的特异性MCO，其含有His和N-末端Asp残基。对修饰肽的LC-MS检测允许我们鉴定抑制A β（1-6）的MCO的抗氧化剂。模型肽的MCO被姜黄素抑制，但不被二丁基羟基甲苯、胡萝卜素、生育酚、雌二醇或尼古丁抑制，这揭示了姜黄素和其他抗氧化剂之间的明显差异。这种新的测定法可以允许鉴定抗氧化剂，其保护免受与退行性疾病相关的肽和蛋白质的MCO。
Production of microregional catalytic reactive oxygen species (ROS) by metal-binding amyloid-beta (A beta) peptides mediates the neurotoxicity of Alzheimer's disease, and inhibitors of this activity may be of therapeutic value. No current analytical methods target specific ROS inhibitors produced by metal-binding peptides. We report a screening assay for metal-catalyzed oxidation (MCO) inhibitors based on liquid chromatography-mass spectrometry (LC-MS) with a model N-terminal A beta peptide (A beta(1-6)). When subjected to MCO by Cu(II)/ascorbic acid, singly and doubly charged A beta(1-6) molecules were observed at m/z 729.2 and 364.8 and m/z 685.3 and 343.3, respectively, corresponding to a decrease in mass of 45 and 89 Da compared with the model peptide. In contrast, H2O2 did not modify the A beta(1-6) peptide. Modified peptides were characterized by a specific MCO of A beta(1-6), which contains both His and N-terminal Asp residues. LC-MS detection of the modified peptides allowed us to identify antioxidants, that inhibit MCO of A beta(1-6). MCO of the model peptide was inhibited by curcumin, but not dibutylhydroxytoluene, carotene, tocopherol, estradiol or nicotine, revealing a clear difference between curcumin and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against MCO of peptides and proteins related to degenerative diseases.