Evidence suggests that amyloid β-protein (Aβ) oligomers may be seminal pathogenic agents in Alzheimer's disease (AD). If so, developing oligomer-targeted therapeutics requires an understanding of oligomer structure. This has been difficult due to the instability of these non-covalently associated Aβ assemblies. We previously used rapid, zero-length, in situ chemical cross-linking to stabilize oligomers of Aβ40. These enabled us to isolate pure, stable populations of dimers, trimers, and tetramers and to determine their structure-activity relationships. However, equivalent methods applied to Aβ42 did not produce stable oligomers. We report here that the use of an Aβ42 homologue, [F10, Y42]Aβ42, coupled with sequential denaturation/dissociation and gel electrophoresis procedures, provides the means to produce highly pure, stable populations of oligomers of sizes ranging from dimer through dodecamer that are suitable for structure-activity relationship determination.

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Protein and peptide oligomers are thought to play important roles in the pathogenesis of a number of neurodegenerative diseases. For this reason, considerable effort has been devoted to understanding the oligomerization process and to determining structure-activity relationships among the many types of oligomers that have been described. We discuss here a method for producing pure populations of amyloid β-protein (Aβ) of specific sizes using the most pathologic form of the peptide, Aβ42. This work was necessitated because Aβ oligomerization produces oligomers of many different sizes that are non-covalently associated, which means that dissociation or further assembly may occur. These characteristics preclude rigorous structure-activity determinations. In studies of Aβ40, we have used the method of photo-induced cross-linking of unmodified proteins (PICUP) to produce zero-length carbon-carbon bonds among the monomers comprising each oligomer, thus stabilizing the oligomers. We then isolated pure populations of oligomers by fractionating the oligomers by size using SDS-PAGE and then extracting each population from the stained gel bands. Although this procedure worked well with the shorter Aβ40 peptide, we found that a significant percentage of Aβ42 oligomers had not been stabilized. Here, we discuss a new method capable of yielding stable Aβ42 oligomers of sizes from dimer through dodecamer.