Taylor, A.I.P. orcid.org/0000-0003-3131-3619, Davis, P.J., Aubrey, L.D. et al. (3 more authors) (2023) Simple, reliable protocol for high-yield solubilization of seedless amyloid-β monomer. ACS Chemical Neuroscience, 14 (1). pp. 53-71. ISSN 1948-7193
Abstract
Self-assembly of the amyloid-β (Aβ) peptide to form toxic oligomers and fibrils is a key causal event in the onset of Alzheimer’s disease, and Aβ is the focus of intense research in neuroscience, biophysics, and structural biology aimed at therapeutic development. Due to its rapid self-assembly and extreme sensitivity to aggregation conditions, preparation of seedless, reproducible Aβ solutions is highly challenging, and there are serious ongoing issues with consistency in the literature. In this paper, we use a liquid-phase separation technique, asymmetric flow field-flow fractionation with multiangle light scattering (AF4-MALS), to develop and validate a simple, effective, economical method for re-solubilization and quality control of purified, lyophilized Aβ samples. Our findings were obtained with recombinant peptide but are physicochemical in nature and thus highly relevant to synthetic peptide. We show that much of the variability in the literature stems from the inability of overly mild solvent treatments to produce consistently monomeric preparations and is rectified by a protocol involving high-pH (>12) dissolution, sonication, and rapid freezing to prevent modification. Aβ treated in this manner is chemically stable, can be stored over long timescales at −80 °C, and exhibits remarkably consistent self-assembly behavior when returned to near-neutral pH. These preparations are highly monomeric, seedless, and do not require additional rounds of size exclusion, eliminating the need for this costly procedure and increasing the flexibility of use. We propose that our improved protocol is the simplest, fastest, and most effective way to solubilize Aβ from diverse sources for sensitive self-assembly and toxicity assays.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 The Authors. Published by American Chemical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Alzheimer’s disease; amyloidogenesis; flow fractionation; multiangle light scattering; peptides; protein preparation |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Funding Information: | Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL BB/P002927/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 12 Jan 2023 12:46 |
Last Modified: | 12 Jan 2023 12:46 |
Status: | Published |
Publisher: | American Chemical Society (ACS) |
Refereed: | Yes |
Identification Number: | 10.1021/acschemneuro.2c00411 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:195121 |