title: Screening assay for metal-catalyzed oxidation inhibitors using liquid chromatography-mass spectrometry with an N-terminal beta-amyloid peptide.
abstract: Production of microregional catalytic reactive oxygen species (ROS) by metal-binding amyloid-beta (Abeta) 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 Abeta peptide (Abeta(1-6)). When subjected to MCO by Cu(II)/ascorbic acid, singly and doubly charged Abeta(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 Abeta(1-6) peptide. Modified peptides were characterized by a specific MCO of Abeta(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 Abeta(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.
authors.affiliation: Department of Physical and Analytical Chemistry, School of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya 463-8521, Japan. kinoue@kinjo-u.ac.jp
authors.forename: Koichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Ako
authors.initials: A
authors.lastname: Nakagawa
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Hino
authors.forename: Hisao
authors.initials: H
authors.lastname: Oka
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry*; D002384:Catalysis; D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006861:Hydrogen Peroxide / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008670:Metals / Q000737:chemistry*; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D017382:Reactive Oxygen Species / Q000737:chemistry*
chemical_list: D016229:Amyloid beta-Peptides; D008670:Metals; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide