title: Separation and characterization of aggregated species of amyloid-beta peptides.
abstract: We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Abeta) peptides from their aggregates. From solutions of Abeta(1-40) or Abeta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of Abeta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for Abeta(1-40) and Abeta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the Abeta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the Abeta-selective antibody mAb1C3, where a monomeric Abeta(1-16) peptide was added to the protofibril preparation. Abeta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.
authors.affiliation: School of Chemical Science and Engineering, Division of Analytical Chemistry, Royal Institute of Technology, 100 44 Stockholm, Sweden.
authors.forename: Henning
authors.initials: H
authors.lastname: Wiberg
authors.forename: Patrik
authors.initials: P
authors.lastname: Ek
authors.forename: Frida Ekholm
authors.initials: FE
authors.lastname: Pettersson
authors.forename: Lars
authors.initials: L
authors.lastname: Lannfelt
authors.forename: Asa
authors.initials: A
authors.lastname: Emmer
authors.forename: Johan
authors.initials: J
authors.lastname: Roeraade
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods*; D047468:Immunoprecipitation; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42)