The Profile of Soluble Amyloid β Protein in Cultured Cell Media DETECTION AND QUANTIFICATION OF AMYLOID β PROTEIN AND VARIANTS BY IMMUNOPRECIPITATION-MASS SPECTROMETRY

Abstract To study the metabolism of amyloid β protein (Aβ) in Alzheimer's disease, we have developed a new approach for analyzing the profile of soluble Aβ and its variants. In the present method, Aβ and its variants are immuno-isolated with Aβ-specific monoclonal antibodies. The identities of the Aβ variants are determined by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The levels of Aβ 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 Aβ species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type or mutant human amyloid precursor protein. In addition to human Aβ-(1-40) and Aβ-(1-42), more than 40 different human Aβ variants were identified. Endogenous murine Aβ and its variants were also identified by this approach. The present approach is a new and sensitive method to characterize the profile of soluble Aβ 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 Aβ and Aβ variants.