Application note

Comparison of microfluidic diffusional sizing and ThT spectroscopy for the assessment of α-synuclein amyloid fibril growth

α-Synuclein is an abundantly expressed neuronal protein that contributes to a host of neurological conditions characterised by Lewy body formations, including Parkinson’s and dementia. α-Synuclein aggregation results in insoluble, beta-sheet rich amyloid fibrils, the growth of which is of key interest in studying the development of these diseases.

Results

Seeded aggregation of α-synuclein monomer solutions was initiated in MES and PB buffers at pH 5.6 and 6.5 respectively using sonicated α-synuclein fibrils. Fibril growth in this solution was measured with Microfluidic Diffusional Sizing (MDS) using the FluidityOne and with ThT (Thioflavin T) spectroscopy using a Clariostar plate reader. The resulting size measurements from the Fluidity One and the change in β-sheet quantity from the ThT assay were normalised and plotted against time to map amyloid fibril growth.

Assessment of α-synuclein amyloid fibril growth (figure 1)
Figure 1: Comparison of α-synuclein fibril growth as measured by MDS and ThT Spectroscopy at pH 5.6 and pH 6.5 A 100µm α-synuclein monomer solution was prepared in 20mM MES at pH 5.6 (80µL) and aggregation was seeded by addition of 10 µL of sonicated α-synuclein fibrils. 10 µL of 300 µm ThT was also added for a final concentration of 80 µm α-synuclein monomer in 16 mM MES buffer pH 5.6. The resulting mix was transferred to a 96 well plate in a single 100 µL aliquot and the ThT fluorescence of the plate was measured over a time course of 1400 minutes (red curve in Panels 1 and 2). A second identical solution was prepared in parallel replacing the ThT with 10 µL of MilliQ water. 3 µL aliquots of this solution were diluted to 6 µL with 20 mM MES immediately before loading on a Fluidity MDS Chip. Fourteen repeat measurements were taken over five hours with a final reading taken at 1400 minutes (Panel 1). This experiment was repeated with 20 mM PB buffer at pH 6.5 in place of 20 mM MES with all other variables maintained between experiments (Panel 2). The second to last measurement in PB buffer was taken after six hours. The data points from these measurements were used to generate the exponential fit present in both panels.

The curves plotted from both data sets are highly comparable indicating MDS data represents a viable alternative to ThT spectroscopy for the measurement of α-synuclein amyloid fibril growth. However, MDS provides some advantages over ThT spectroscopy for this application. The ThT label is bound to the β-sheets of the fibril throughout the experiment and may affect the behaviour of the protein during measurement. MDS measures the protein in solution, in its native state and labelling only occurs following diffusion, preventing the label from influencing the measurement. Furthermore, some amyloid fibrils are not detected by ThT fluorescence and can provide false negative results. MDS has the capability to detect these fibrils due to its latent labelling system and direct detection of the size of protein species.

MDS represents a new approach for studying aggregation of α-synuclein and other proteins in their native state. Further to this, MDS bypasses many of the limitations of alternative techniques, such as requirement for surface functionalisation (SPR) or matrix separation (SEC), providing a new experimental option for researchers.

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In-solution sizing and quantification of native protein in less than 10 minutes

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