The profile of soluble amyloid beta protein in cultured cell media - Detection and quantification of amyloid beta protein and variants by immunoprecipitation mass spectrometry

To study the metabolism of amyloid beta protein (A beta) in Alzheimer's disease, we have developed a new approach for analyzing the profile of soluble A beta and its variants, In the present method, A beta and its variants are immunoisolated with A beta-specific monoclonal antibodies. The identities of the Ap variants are determined by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The levels of A beta variants are determined by their relative peak intensities in mass spectrometric measurements by comparison with internal standards of known identities and concentrations. We used this method to examine the A beta species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type or mutant human amyloid precursor protein. In addition to human A beta-(1-40) and A beta-(1-42), more than 40 different human A beta variants were identified. Endogenous murine A beta and its variants were also identified by this approach. The present approach is a new and sensitive method to characterize the profile of soluble A beta in conditioned media and biological fluids. Furthermore, it allows direct measurement of each individual peptide in a peptide mixture and provides comprehensive information on the identity and concentration of A beta and A beta variants.