Screening assay for metal-catalyzed oxidation inhibitors using liquid chromatography-mass spectrometry with an N-terminal beta-amyloid peptide.
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.
Inoue
Koichi
K
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
Nakagawa
Ako
A
Hino
Tomoaki
T
Oka
Hisao
H
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*
D016229:Amyloid beta-Peptides; D008670:Metals; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide