α-Synuclein purification significantly impacts seed amplification assay performance and consistency

-Synuclein seed amplification assays are a promising diagnostic tool for synucleinopathies such as Parkinsons disease and multiple system atrophy. Standardized conditions are required to ensure a high degree of inter- and intra-laboratory reproducibility when performing these assays. A significant issue that hinders the utility of seed amplification assays is the de novo aggregation propensity of the -synuclein substrate as well as inter-batch heterogeneity. While much work has focused on determining appropriate seed amplification assay buffer compositions as well as the type and amount of seed used, a robust comparison of -synuclein substrate purification methods has not been reported. We therefore compared the utility of recombinant -synuclein purified using four different methods as seed amplification assay substrates across two laboratories. Osmotic shock-purified -synuclein monomer substrate showed the lowest propensity for de novo aggregation, which translated into being the best substrate for seed amplification assay reactions seeded with -synuclein preformed fibrils or patient brain homogenates. Furthermore, osmotic shock -synuclein monomer showed the best inter-batch reproducibility compared to all other substrates tested. As -synuclein seed amplification assays continue to evolve and move towards adoption in the clinical realm, this work showcases the vital importance of standardizing the production and characterization of recombinant -synuclein substrate. We encourage the widespread adoption of osmotic shock -synuclein monomer as the universal substrate for seed amplification assays to maximize intra- and inter-laboratory reproducibility.