TITLE

Separation and characterization of aggregated species of amyloid-beta peptides

AUTHOR(S)
Wiberg, Henning; Ek, Patrik; Pettersson, Frida Ekholm; Lannfelt, Lars; Emmer, Åsa; Roeraade, Johan
PUB. DATE
July 2010
SOURCE
Analytical & Bioanalytical Chemistry;Jul2010, Vol. 397 Issue 6, p2357
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
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 Aβ1–40 or Aβ1–42 monomeric peptides, low molecular weight material appeared at a p I value of ca. 5, while the presence of aggregates was detected as bands, observed at a p I 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 Aβ1–40 and Aβ1–42, 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 Aβ1–42 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 Aβ1–16 peptide was added to the protofibril preparation. Aβ1–16 is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.
ACCESSION #
51880441

 

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