In vitro Aggregation Ability of Five Commercially Available Aβ42 peptide

Background: 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. Objective: To analyze the consistency of the aggregation ability of 5 commercially available Aβ42 peptide. Methods: 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. Results: 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. Conclusion: 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.