Simple and Novel Screening Assay of Natural Antioxidants for Cu(II) Ion/Adrenaline-Mediated Oxidation of N-Terminal Amyloid β by Liquid Chromatography/Mass Spectrometry

Abstract

In the present study, a novel assay for screening inhibitors of Cu(II) ion/adrenaline-mediated oxidative modification of N-terminal amyloid beta (Abeta) peptides was developed using liquid chromatography with mass spectrometry (LC/MS). The physiological condition of Cu(II) ion/adrenaline in buffer (pH 7.4) at 37 degrees C for 90 min revealed a specific modification of N-terminal Abeta peptides, such as Abeta1-6, Abeta1-40, and Abeta1-42, using trypsin digestion and LC/MS detection of the modified Abeta peptide. When this oxidative modification of the shorter N-terminal Abeta1-6 was subjected to LC/MS, single charged ions from native peptide ([M+H]+, m/z 774) were observed at m/z 729 and 685, corresponding to a decrease in mass of 45 and 89 Da, respectively, as compared with the original peptide. To determine the effect of specific antioxidants, a screening assay to find inhibitors of Cu(II) ion/adrenaline-mediated oxidation was developed based on the response ratio of m/z 685 to 774. LC/MS detection of the modified peptides allowed us to identify antioxidants that inhibit oxidative modification of Abeta1-6 model peptide. The oxidative modification of Abeta1-6 was inhibited by curcumin but not an isoflavone or catechin mixture or saponin or capsaicin, revealing a clear difference between antioxidants that inhibit oxidative modification and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against oxidative modification of Abeta and other proteins related to oxidative stress by adrenaline and Cu(II) ions under normal physiologic conditions.