Preparation and NMR characterization of Aβ peptides at pathological pH

Alzheimer's disease (AD) is a neurodegenerative disorder marked by the progressive deterioration of cognitive function. Its pathological hallmarks include the formation of amyloid plaques, which are primarily due to the abnormal aggregation of Aβ peptides. However, the propensity of Aβ peptides for aggregation makes the in vitro preparation very challenging, often resulting in low yield, instability, and impurities. Here in this study, we developed an in vitro method for preparing monomeric Aβ peptides to achieve stable and high-purity samples. Specifically, three strategies including the uses of high concentration of protein denaturant urea, alkaline buffer (ammonium carbonate buffer), and organic solvents (acetonitrile, hexafluoroisopropanol) were applied to prevent Aβ aggregation during the purification. Through an optimized production process, we successfully obtained stable and highly pure ¹⁵N, ¹³C-doubly labeled monomeric Aβ40 and Aβ42 peptides suitable for NMR data collection at the pathological acidic pH. Overall, the preparation method presented here offer a robust approach for in vitro production of monomeric Aβ peptides with satisfying purity and reproducibility. Meanwhile, the NMR characterization results for Aβ40 and Aβ42 at pH 6.5 provide useful information for the further biophysical studies involving these two peptides.