Mass spectrometry-based screening for inhibitors of β-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 (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 degrees 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.