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 (Abeta) in Alzheimer's disease, we have developed a new approach for analyzing the profile of soluble Abeta and its variants. In the present method, Abeta and its variants are immuno-isolated with Abeta-specific monoclonal antibodies. The identities of the Abeta variants are determined by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The levels of Abeta 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 Abeta species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type or mutant human amyloid precursor protein. In addition to human Abeta-(1-40) and Abeta-(1-42), more than 40 different human Abeta variants were identified. Endogenous murine Abeta and its variants were also identified by this approach. The present approach is a new and sensitive method to characterize the profile of soluble Abeta 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 Abeta and Abeta variants.
Wang
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R
Laboratory for Mass Spectrometry, The Rockefeller University, New York, New York 10021, USA.
Sweeney
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D
Gandy
S E
SE
Sisodia
S S
SS
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D000818:Animals; D003470:Culture Media; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014407:Tumor Cells, Cultured
D016229:Amyloid beta-Peptides; D003470:Culture Media; D010450:Endopeptidases