In vitro Aggregation Ability of Five Commercially Available Aβ₄₂ peptide

As the most basic material, synthetic human Amyloid-β (1-42) (Aβ₄₂) peptide from different manufacturers have been widely used. Their aggregation ability is vital to the reliability, repeatability and comparability of studies on Aβ₄₂ physiology and pathology. However, it has not been evaluated and compared.

To analyze the consistency of the aggregation ability of 5 commercially available Aβ₄₂ peptide.

5 Aβ₄₂ peptide represented as A, B, C, D and E were pretreated by HFIP. The pretreated Aβ₄₂ peptide were dissolved in Thioflavin T (ThT) solution, and their aggregation kinetics was monitored for 30 h with the aggregation kinetics test. Meanwhile, the pretreated peptide were aggregated in phosphate buffered saline. After aggregated for 12 h, they were detected by methods of ThT fluorescence, far-UV circular dichroism (CD), SDS-PAGE, western blot, and transmission electron microscopy (TEM), respectively. After aggregation for 8 h and 12 h, their cytotoxicity to SH-SY5Y cells was further evaluated using Cell Counting Kit-8.

For aggregation kinetics, peptide A, C and E remained low level curves, while peptide B and D presented typical sigmoidal kinetics curves. In CD measurement, the aggregates of peptide B and D showed relatively high negative CD peaks with the height of -8.09 mdeg and -14.37 mdeg, while the height of peptide A, C and E was -1.04, -3.55, and -3.88. In ThT assay, relative fluorescence intensity of the aggregates of peptide B and D were 7.79 and 8.82, higher than 1.19, 1.71, and 2.70 of peptide A, C and E, respectively. In SDS-PAGE, all aggregates contained monomers and eleven polymers. Moreover, peptide B-E presented a trapezoidal distribution from dimers to trimers, and peptide A aggregated to dimers. By western blot, the bands of monomers remained in all aggregates. Furthermore, peptide B and D aggregated to dimers and trimers, peptide A and C only aggregated to dimers, and peptide E showed a strong band of trimers. By TEM, protofibrils were observed only in peptide B, while substantial spherical aggregates were formed in other peptide. Additionally, peptide B, D and E exhibited higher cytotoxicity after aggregated for 8 h, whereas peptide A, B and D presented relatively high cytotoxicity after 12-hour aggregation.

Commercially available Aβ₄₂ peptide showed obvious differences in aggregation ability, which should arouse enough attention in the field of basic study related to Aβ₄₂. The aggregation ability evaluation with the various assay methods has some discrepancies, and it is highly urgent to establish a reasonable and uniform measurement strategy.