Mass Spectrometry-Based Screening for Inhibitors of beta-Amyloid Protein Aggregation

摘要

Alzheimer's disease is the most common cause of the loss of cognitive function among the elderly, and the aggregation and deposition of misfolded beta-amyloid protein (A(beta)) contribute to this progressive central nervous system decline. Therefore, compounds that inhibit or even reverse A(beta) aggregation might be useful for the treatment or prevention of Alzheimer's disease. To identify potential therapeutic agents for the treatment of Alzheimer's disease, a mass spectrometry-based screening assay was developed to identify and rank order compounds that inhibit the aggregation of A(beta). To carry out this assay, A(beta) was incubated with a test compound at 37 deg C for 20 h followed by ultrafiltration to separate the monomeric A(beta) from its aggregates. Aliquots of the ultrafiltrate were analyzed for monomeric A(beta) using positive ion electrospray mass spectrometry based on the abundance the quadruply protonated molecule of A(beta) at m/z 1083. The calibration curve for A(beta) was linear with a correlation coefficient (r~(2)) of >0.99 over the range of at least 11-110 (mu)M. The limit of detection was 0.224 ng (5.18 nM, 10-(mu)L injection), and the limit of quantitation was 0.747 ng (17.2 nM, 10-(mu)L injection). Based on previous reports of compounds that either bind to A(beta) or are useful in treating Alzheimer's disease, melatonin, methysticin, 3-indolepropionic acid, and daunomycin were assayed and ranked in order of inhibition of A(beta) aggregation. The most effective inhibitor of aggregation of A(beta) protein was daunomycin followed in descending order by 3-indolepropionic acid, melatonin, and then methysticin. These data suggest that this ultrafiltration LC-MS screening assay may be used to identify potential therapeutic agents for the treatment of Alzheimer's disease based on the prevention of A(beta) aggregation.