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Separation and characterization of aggregated species of amyloid-beta peptides
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
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2010 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 397, no 6, 2357-2366 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (A beta) peptides from their aggregates. From solutions of A beta(1-40) or A beta(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 A beta 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 A beta(1-40) and A beta(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 A beta(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 A beta-selective antibody mAb1C3, where a monomeric A beta(1-16) peptide was added to the protofibril preparation. A beta(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.

Place, publisher, year, edition, pages
2010. Vol. 397, no 6, 2357-2366 p.
Keyword [en]
Alzheimer's disease, Protofibrils, Amyloid-beta (A beta), Isoelectric focusing (IEF)., Mass spectrometry (MS), Bioanalytical methods, Capillary electrophoresis / Electrophoresis
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-136871DOI: 10.1007/s00216-010-3839-9ISI: 000279453000038PubMedID: 20502882OAI: oai:DiVA.org:uu-136871DiVA: diva2:377880
Available from: 2010-12-15 Created: 2010-12-14 Last updated: 2017-12-11Bibliographically approved

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Ekholm Pettersson, FridaLannfelt, Lars

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