Separation and characterization of aggregated species of amyloid-beta peptides

We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Aβ) peptides from their aggregates. From solutions of Aβ_1–40 or Aβ_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 Aβ 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 Aβ_1–40 and Aβ_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 Aβ_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 Aβ-selective antibody mAb1C3, where a monomeric Aβ_1–16 peptide was added to the protofibril preparation. Aβ_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.