Protocol for quantifying the oxidative cleavage of amyloid-β using oxygen-18 isotope labeling and LC-MS/MS

Here, we present a protocol for quantifying the oxidative cleavage of amyloid-β using oxygen-18 labeling coupled with high-resolution LC-MS/MS. We describe procedures for amyloid-β peptide preparation, copper(II)/ascorbate-mediated oxidative cleavage, oxygen-18 incorporation, LC-MS/MS acquisition, and isotopologue-based quantitative data analysis. By incorporating oxygen-18 into newly generated C-termini during metal-driven cleavage and applying defined isotope conditions, this protocol enables site-specific identification of true cleavage-derived fragments while discriminating them from background signals arising from natural isotopic overlap and pre-existing impurities. This strategy supports confident identification of oxidative cleavage products and can be adapted to studies of peptide degradation in related systems. For complete details on the use and execution of this protocol, please refer to Yang et al.¹.