Mass spectrometry-based screening for inhibitors of beta-amyloid protein aggregation.

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 (Abeta) contribute to this progressive central nervous system decline. Therefore, compounds that inhibit or even reverse Abeta 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 Abeta. To carry out this assay, Abeta was incubated with a test compound at 37 degrees C for 20 h followed by ultrafiltration to separate the monomeric Abeta from its aggregates. Aliquots of the ultrafiltrate were analyzed for monomeric Abeta using positive ion electrospray mass spectrometry based on the abundance the quadruply protonated molecule of Abeta at m/z 1083. The calibration curve for Abeta was linear with a correlation coefficient (r2) of >0.99 over the range of at least 11-110 microM. The limit of detection was 0.224 ng (5.18 nM, 10-microL injection), and the limit of quantitation was 0.747 ng (17.2 nM, 10-microL injection). Based on previous reports of compounds that either bind to Abeta or are useful in treating Alzheimer's disease, melatonin, methysticin, 3-indolepropionic acid, and daunomycin were assayed and ranked in order of inhibition of Abeta aggregation. The most effective inhibitor of aggregation of Abeta 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 Abeta aggregation.