Screening Assay for Metal-Catalyzed Oxidation Inhibitors Using Liquid Chromatography-Mass Spectrometry with an N-Terminal beta-Amyloid Peptide

摘要

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, H_(2)O_(2) 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.