Method for rapid preparation of multi-sized alpha-synuclein aggregates

Abstract

The application provides a method for rapidly preparing multi-size alpha-synuclein aggregates, and solves the problems of complicated steps and high cost in the prior art method for preparing alpha-synuclein aggregates of different sizes. The method comprises the following steps: 1) dissolving and uniformly mixing the separated and purified alpha-synuclein monomers in a physiological buffer solution to obtain a monomer protein solution; 2) placing the monomer protein solution in a centrifugal tube, and shaking and incubating under certain conditions, and sampling at different time points in the incubation process; 3) adding the samples into ultrafiltration centrifugal tubes, and centrifuging and separating to remove small-size proteins; 4) adding a physiological buffer solution into the ultrafilters of the ultrafiltration centrifugal tubes, and inverting the ultrafilters into centrifugal tubes matched with the ultrafilters, centrifuging and separating to collect the protein aggregates on the membranes in the ultrafilters, and obtaining protein aggregates of different morphological structures.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and new medicine, specifically relating to a rapid preparation method for multi-sized α-synuclein aggregates. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by motor and non-motor symptoms such as bradykinesia, tremor, constipation, mental disorders, and sleep disturbances. These clinical symptoms significantly impact the daily lives of PD patients and impose immense pressure on society. Despite remarkable progress in understanding its pathogenesis and epidemiology in recent years, the cause of Parkinson's disease remains a mystery, and no cure or preventative treatment has yet been found. The most important pathological feature of Parkinson's disease is the extensive degeneration and death of dopaminergic neurons in the substantia nigra pars compacta of the brain, leading to a significant reduction in striatal dopamine levels, which is associated with motor symptoms such as bradykinesia and tremor. During the disease progression, distinct pathological features appear in the limbic system and cortex of the brain, and are associated with a range of non-motor characteristics.

[0003] Alpha-synuclein (α-Syn) is a 140-residue protein widely expressed in the brain, interacting with many specific proteins involved in signal transduction, vesicle transport, synaptic behavior, oxidative stress regulation, and mitochondrial function. It is a major component of protein-rich inclusion bodies, Lewy bodies, and Lewy neurites—the hallmark lesions of Parkinson's disease (PD). Throughout the development of PD, α-synuclein monomers transform over time into proteins of different sizes, structures, and morphologies. The coexistence of all these different α-synucleins makes identifying the toxic species in the body challenging. Recent research has increasingly shown that soluble oligomers of α-synuclein play a crucial role in the development of PD. While there is no clear definition of oligomers, they include unstable soluble aggregates existing between monomers and insoluble filaments. The mechanisms by which oligomers severely lead to neuronal degeneration and death include cell membrane disruption, synaptic damage, mitochondrial dysfunction, and neuroinflammation.

[0004] Therefore, the precise preparation of α-synuclein oligomers of different sizes (i.e., different molecular weights and morphologies) is of great significance for the study of the occurrence and development of Parkinson's disease (PD). Lashuel et al. used liquid chromatography to purify α-synuclein oligomers of different sizes and observed their morphological differences. Emin et al. used the mechanism that proteins accumulate at different masses in different concentrations of sucrose during centrifugation to prepare a series of α-synuclein aggregates of different sizes using the sucrose gradient method. However, the above-mentioned α-synuclein oligomer preparation and purification methods are relatively cumbersome (for example, the introduction of impurities such as sucrose adds subsequent steps to remove these impurities) and require expensive equipment such as protein liquid chromatography. Currently, there is still a lack of low-cost, efficient, rapid and simple methods for preparing α-synuclein aggregates.

[0005] Therefore, this invention explores a rapid preparation method for multi-sized α-synuclein aggregates. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing methods for preparing α-synuclein aggregates of different sizes, which are cumbersome and costly, and to provide a rapid method for preparing multi-sized α-synuclein aggregates that is both simple and efficient.

[0007] To achieve the above objectives, the technical solution provided by this invention is:

[0008] A rapid method for preparing multi-sized α-synuclein aggregates, characterized by the following steps:

[0009] 1) Dissolve the isolated and purified α-synuclein monomers in PBS buffer solution at 2-6℃ according to the concentration standard of 0.8-1.2 mg / ml (only physiological buffer solution can be used, otherwise other factors will be introduced to affect protein aggregation) and mix well (you can use vortexing, inversion, pipetting, etc. to help dissolve the α-synuclein monomers and mix well) to obtain monomer protein solution;

[0010] 2) Place the monomeric protein solution obtained in step 1) into a centrifuge tube and incubate it at 300-700 rpm under human physiological temperature (shaking incubation can accelerate the protein aggregation speed and make the generated aggregates more uniform in size). Take samples at different time points during the incubation process (several time points can be selected according to the required protein size and the particle size change pattern, i.e., different sampling times can be selected for different needs). Each sample should be no less than 200 μL (considering that there will be protein loss during ultrafiltration centrifugation and re-centrifugation collection, if the sample volume is too small, the final protein aggregate concentration will be low. Therefore, the sample volume in this step should be no less than 200 μL).

[0011] 3) Add the samples obtained in step 2) to 100kDa ultrafiltration centrifuge tubes (because the molecular weight of α-synuclein monomers is 14kDa, using this type of ultrafiltration centrifuge tube can ensure that α-synuclein monomers pass through the filter membrane, while effectively retaining aggregates on the filter membrane), and centrifuge at 2-6℃ and 14000-16000g for 3-10min to separate the monomers from the aggregates. At this time, what remains in the ultrafiltration centrifuge tube is the protein aggregate;

[0012] 4) Add 50-100 μL of PBS buffer to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into the centrifuge tube it is paired with. Centrifuge at 2-6℃ and 300-600g for 3-10 min. Collect the protein aggregates on the inner membrane of each ultrafilter. At this time, protein aggregates of different sizes (i.e., different morphological structures and different molecular weights) can be obtained and stored at 2-6℃ for later use.

[0013] Therefore, this invention can obtain α-synuclein aggregates of different sizes after incubation of the same α-synuclein monomer for different times. The size of the protein aggregates can be controlled by adjusting the incubation time throughout the preparation process. This invention avoids introducing protein aggregates of different sizes by separating at different incubation time points, reducing the difficulty of separation and purification. Furthermore, the protein aggregates obtained from separation and purification at different incubation levels exhibit good size uniformity, yielding protein aggregates and protein fibers of varying sizes, including small, medium, and large. Characterization also confirms that the aggregates prepared by this method are relatively uniform in size, rather than being a mixture of various aggregates, demonstrating the simplicity, speed, and efficiency of this preparation method.

[0014] Further, in step 1), the isolated and purified α-synuclein monomers are dissolved in PBS buffer at 4°C (the monomers are more stable at 4°C) according to the concentration standard of 1 mg / ml, and vortexed to mix to obtain a monomeric protein solution.

[0015] Further, in step 2), the monomeric protein solution obtained in step 1) is placed in a centrifuge tube and incubated at 37°C and 500 rpm with shaking. Samples are taken after 6 h, 15 h, 24 h, and 48 h of incubation, and each sample is no less than 200 μL. Depending on the requirements, sampling, separation, and purification can be performed at the above incubation time points to obtain protein aggregates with different morphological structures and significantly different sizes.

[0016] Further, in step 3), the samples obtained in step 2) are added to 100kDa ultrafiltration centrifuge tubes and centrifuged at 4°C and 15300g for 5 minutes to separate the monomers from the aggregates.

[0017] Further, in step 4), protein aggregates with different morphologies on the inner membrane of the ultrafilter are collected by centrifugation at 4°C and 450g for 4 min and stored at 4°C for later use.

[0018] Meanwhile, the present invention uses the above-mentioned rapid preparation method to prepare α-synuclein aggregates of various sizes with uniform morphology by sampling at different incubation times.

[0019] The multi-sized α-synuclein aggregates obtained by the rapid preparation method of this invention can be applied to the pathological research of α-synucleinosis, such as Parkinson's disease. Specifically, the method for studying Parkinson's disease pathology involves differentiating neuroblastoma cells (SH-SY5Y cells) into mature dopaminergic neurons and inducing Parkinson's disease pathology. The specific differentiation method uses a combination of retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) to differentiate SH-SY5Y cells into mature dopaminergic neurons. Alternatively, the degree of damage to the blood-brain barrier caused by α-synuclein aggregates of different morphologies can be observed in a Parkinson's disease microarray model with blood-brain barrier function. Based on the above applications, the α-synuclein aggregates obtained by the above rapid preparation method can also be used to induce α-synucleinosis, such as Parkinson's disease. In cultured mature dopaminergic neurons, exogenous α-synuclein monomers or aggregates of different morphologies can be added to reproduce the abnormal aggregation of α-synuclein, oxidative stress, and mitochondrial dysfunction in Parkinson's disease.

[0020] The advantages of this invention are:

[0021] 1. The preparation method of this invention only requires ultrafiltration centrifuge tubes, combined with controlled shaking incubation time, to prepare and extract α-synuclein aggregates of different sizes, greatly reducing the preparation cost of α-synuclein aggregates. The preparation method is simple, efficient, and yields stable and uniform protein aggregates. Furthermore, based on this method, neurotoxicity analysis of α-synuclein aggregates can also be performed, confirming that medium-sized α-synuclein aggregates have the strongest neuronal and mitochondrial toxicity, providing a powerful tool for research on the occurrence and development of Parkinson's disease.

[0022] 2. By controlling the incubation time using the method of this invention, protein aggregates of different sizes can be prepared. The entire preparation method is ingeniously conceived, fast and simple, greatly simplifying the preparation process. Moreover, the prepared protein aggregates are relatively stable and uniform. Furthermore, no expensive equipment is used in the preparation process, which significantly reduces the preparation cost.

[0023] 3. This invention also characterized and confirmed the target product using various physical and biological methods. The prepared α-synuclein aggregates can be used not only for pathological research and the establishment of in vitro disease models for Parkinson's disease, but also for pathological research and model establishment for other α-synuclein diseases such as Lewy body dementia and multiple system atrophy. Furthermore, the prepared α-synuclein aggregates can be used in organ-on-a-chip to explore the interaction between the disease and other organs, such as investigating the damage to the blood-brain barrier caused by Parkinson's disease. All of these applications further demonstrate the practicality of the α-synuclein aggregates with different morphologies prepared by this invention. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the process for extracting and purifying α-synuclein monomers according to the present invention;

[0025] Figure 2 This is a schematic diagram illustrating the preparation of α-synuclein aggregates with different morphologies and structures according to the present invention;

[0026] Figure 3 TEM images of α-synuclein monomers and α-synuclein aggregates with different morphologies of the present invention; a. α-synuclein monomer (M); b. Oligomer 1 (O1, 6h); c. Oligomer 2 (O2, 15h); d. Short filament (SF, 24h); e. Long filament (LF, 48h); enlarged view of a portion in fc;

[0027] Figure 4 AFM characterization diagrams of α-synuclein monomers and α-synuclein aggregates with different morphologies of the present invention are shown below: a. α-synuclein monomer (M); b. Oligomer 1 (O1, 6h); c. Oligomer 2 (O2, 15h); d. Short filament (SF, 24h); e. Long filament (LF, 48h).

[0028] Figure 5 Bright-field imaging images of different morphological structures of SH-SY5Y cells before and after differentiation in this invention (scale bar: 50 μm);

[0029] Figure 6 This invention provides a characterization of differentiated and mature dopaminergic neurons.

[0030] Figure 7 To characterize the differentiated mature dopaminergic neurons of this invention, the differentiated mature dopaminergic neurons significantly express dopamine transporter (DAT) and tyrosine hydroxylase (TH);

[0031] Figure 8 The changes in mitochondrial membrane potential levels after treatment of differentiated mature dopaminergic neurons with α-synuclein aggregates of different morphologies and structures according to the present invention.

[0032] Figure 9 The changes in DAT expression levels after treatment of differentiated mature dopaminergic neurons with α-synuclein aggregates of different morphologies according to the present invention (scale bar: 100 μm);

[0033] Figure 10 For the quantitative analysis of DAT protein expression level fluorescence morphology in this invention; a. average area; b. average branch length; c. average fluorescence intensity; d. average network number;

[0034] Figure 11 The changes in TH expression levels in differentiated mature dopaminergic neurons after treatment with α-synuclein aggregates of different morphologies according to the present invention (scale bar: 100 μm);

[0035] Figure 12 For the quantitative analysis of TH protein expression level fluorescence morphology in this invention; a. average area; b. average branch length; c. average fluorescence intensity; d. average network number;

[0036] Figure 13 The levels of reactive oxygen species (ROS) produced after treatment of differentiated mature dopaminergic neurons with α-synuclein aggregates of different morphologies according to the present invention (scale bar: 200 μm).

[0037] Figure 14 For the morphological quantitative analysis of the ROS-generated horizontal fluorescence pattern of this invention, the following values ​​are used: a. average area; b. average branch length; c. average fluorescence intensity; d. average network number. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0039] This invention first extracts α-synuclein monomers, and then uses shake-incubation and ultrafiltration to prepare a series of α-synuclein aggregates with different morphologies for inducing Parkinson's disease. The prepared α-synuclein aggregates act on differentiated mature dopaminergic neurons. By detecting the expression levels of marker proteins, the production levels of reactive oxygen species, and the damage levels of mitochondrial membrane potential, the most neurotoxic α-synuclein aggregate was identified. Cellular-level experiments validated the identification of an α-synuclein aggregate with the greatest impact on nerve cells.

[0040] The specific preparation method is as follows:

[0041] 1) Extraction and purification of α-synuclein monomers, such as... Figure 1 As shown

[0042] 1.1) The recombinant α-synuclein Escherichia coli strain was constructed by the research group in the previous stage and cultured at 37℃ with shaking until the OD600 reached 0.6-0.8.

[0043] 1.2) After adding IPTG (isopropyl-β-D-thiogalactoside), the mixture was induced at 37℃ for 4 h, and the fermentation broth was centrifuged at 15000 rpm for 10 min to collect the cells.

[0044] 1.3) After disrupting the bacterial cells using an ultrasonic disruptor, centrifuge the mixture, collect the supernatant, and purify the impurity protein solution using a nickel column to obtain α-synuclein with a purity of over 95%.

[0045] 1.4) The purified α-synuclein was freeze-dried in a freeze dryer and stored at -20°C for a long time.

[0046] 2) Preparation of α-synuclein aggregates with different morphologies and structures, the specific preparation process is as follows: Figure 2 As shown:

[0047] 2.1) Dissolve the obtained α-synuclein monomer in PBS buffer at 4°C to 1 mg / ml and vortex to obtain a monomeric protein solution.

[0048] 2.2) Place the obtained monomeric protein solution in a 2 ml centrifuge tube and incubate at 37°C and 500 rpm with shaking.

[0049] 2.3) Samples were taken at 6h, 15h, 24h and 48h of incubation, and each sample was no less than 200μL.

[0050] 2.4) The sampled solutions were added to 100kDa ultrafiltration centrifuge tubes and centrifuged at 4℃ and 15300g for 5min to separate monomers from aggregates and obtain proteins with small sizes removed.

[0051] 2.5) Add 100 μL of PBS buffer to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into the centrifuge tube it is used with. Centrifuge at 4℃ and 450g for 4 min to collect protein aggregates on the inner membrane of each ultrafilter. Finally, protein aggregates with different morphologies and structures are obtained and stored at 4℃ for later use.

[0052] 3) Characterization of α-synuclein aggregates with different morphologies

[0053] 3.1) Transmission electron microscopy (TEM) characterization

[0054] The morphology of the prepared α-synuclein aggregates was characterized using transmission electron microscopy (TEM). The sample preparation method for TEM samples is as follows:

[0055] ① Dilute the sample with ultrapure water to 0.1-1 μM;

[0056] ② Drop the sample solution directly onto a copper grid with a supporting membrane and let it stand for 5 minutes;

[0057] ③ Use filter paper strips to absorb excess liquid from the edge of the droplet and allow it to dry slightly;

[0058] ④ Add 5% phosphotungstic acid staining solution for negative staining and let stand for 3 minutes;

[0059] ⑤ Use filter paper strips to absorb excess dye, dry at 37℃ for 8 hours, and then observe using a transmission electron microscope.

[0060] TEM images of α-synuclein aggregates with different morphologies and structures prepared using this method are shown below. Figure 3 According to the TEM images, the particle size of the protein aggregates gradually increases with the increase of shaking incubation time. Two types of α-synuclein oligomers with different particle sizes are formed at 6h and 15h of shaking incubation, while short fibers are observed at 24h and long fibers are visible at 48h, showing obvious size changes.

[0061] 3.2) Atomic force microscopy (AFM) characterization

[0062] The morphology of the prepared α-synuclein aggregates was characterized using atomic force microscopy (AFM). The sample preparation method for AFM is as follows:

[0063] ① Use ultrapure water to dilute the concentration of α-synuclein to about 50 μg / ml;

[0064] ② Pipette 40-50 μL of sample solution onto a clean mica sheet and let it stand for 5-10 minutes to allow the sample to deposit onto the surface of the mica sheet;

[0065] ③ Add a small amount of ultrapure water to wash away the PBS contained in the sample;

[0066] ④ Dry at room temperature until there are no obvious water marks on the surface before using it for imaging analysis.

[0067] AFM images of α-synuclein aggregates with different morphologies prepared using this method are shown below. Figure 4 As shown, AFM morphology imaging of different α-synuclein aggregates and Figure 3TEM results showed excellent agreement. Before fiber formation, the particle size of α-synuclein aggregates tended to increase with prolonged shaking incubation time, yielding two different particle sizes of oligomers at 6 h and 15 h of shaking incubation. Similarly, samples incubated for 24 h and 48 h exhibited short and long fiber morphologies, respectively. Furthermore, it is worth noting that AFM results showed an increasing longitudinal height of α-synuclein with increasing shaking incubation time.

[0068] 4) Inducing Parkinson's disease

[0069] 4.1) Specific implementation methods for dopaminergic neuron differentiation:

[0070] SH-SY5Y cells were first differentiated for 3 days in DMEM complete medium containing 20 μM RA (with 10% FBS and 1% P / S) in culture flasks. Then, the cells were collected and resuspended in DMEM medium containing BDNF and low serum (with 2.5% FBS and 1% P / S). The suspension was then seeded into wells of plates or microarray channels for further differentiation for 3 days. Ultimately, SH-SY5Y cells successfully differentiated into dopaminergic neurons. The morphology of the differentiated cells was observed using a fluorescence microscope. Figure 5 As shown in the image (the morphology of the cells in the image shows that SH-SY5Y cells have successfully differentiated into dopaminergic neurons), and quantitative morphological analysis was performed on the bright-field imaging images of SH-SY5Y cells before and after differentiation, as follows: Figure 6 Analysis showed that the branch length of differentiated cells was significantly increased compared to that before differentiation. Furthermore, after differentiation induced by a combination of RA and BDNF, immunofluorescence staining and laser confocal microscopy revealed that SH-SY5Y cells significantly expressed dopaminergic neuronal marker proteins such as dopamine transporter (DAT) and tyrosine hydroxylase (TH). Figure 7 This also proves that SH-SY5Y cells have successfully differentiated into dopaminergic neurons.

[0071] 4.2) Specific methods for α-synuclein-induced Parkinson's disease

[0072] For the differentiated dopaminergic neurons, the medium was replaced with low-serum (2.5% FBS) DMEM containing 4.2 μg / ml α-synuclein aggregates of different morphologies to induce Parkinson's disease pathology. The medium was changed every 24 hours for 48 hours, at which point the induction of Parkinson's pathology was completed.

[0073] 4.3) Symptoms of Parkinson's Disease Induction

[0074] 4.3.1) Specific implementation method of mitochondrial membrane potential probe:

[0075] JC-10 is a fluorescent probe capable of rapidly and sensitively detecting changes in mitochondrial membrane potential in cells or tissues, and is commonly used for early detection of apoptosis. When the mitochondrial membrane potential is high, JC-10 aggregates in the mitochondrial matrix, forming polymers that produce red fluorescence; when the mitochondrial membrane potential is low, JC-10 cannot aggregate in the mitochondrial matrix, and remains a monomer, producing green fluorescence. The JC-10 fluorescent probe was used to detect changes in mitochondrial membrane potential before and after treatment with α-synuclein aggregates of different morphologies, assessing mitochondrial dysfunction. Cells were stained with 2.5 μM JC-10, and flow cytometry was used to detect the red-green fluorescence ratio and calculate the results.

[0076] The results are as follows Figure 8 As shown, compared with the control group, the red fluorescence of mitochondria was weakened and the green fluorescence was enhanced in cells treated with α-synuclein aggregates of different morphologies, indicating that the mitochondrial activity of the cells was reduced. This shows that α-synuclein aggregates impaired the mitochondrial function of the cells. The red-green fluorescence ratio was significantly reduced after treatment with oligomers, and oligomer 2 (O2) severely impaired the mitochondrial membrane potential.

[0077] 4.3.2) Specific implementation method for immunofluorescence staining to detect protein expression levels:

[0078] First, cells were fixed with 4% paraformaldehyde fixative, followed by permeabilization and blocking. Then, differentiated SH-SY5Y cells were incubated with primary antibodies against dopaminergic neuron markers DAT and TH, and Goat anti-rabbit IgG (Alexa) was administered. 488) was used as a secondary antibody for incubation, and imaging was finally performed using laser confocal microscopy. After Parkinson's pathological induction, the degree of decrease in the expression levels of DAT and TH, the hallmark proteins of dopaminergic neurons, was observed to identify the most toxic α-synuclein aggregates. Figure 9 and Figure 11 It can be seen that the expression of DAT and TH was significantly reduced after treatment with oligomer 1 (O1) and oligomer 2 (O2). Furthermore, morphological quantitative analysis was performed on the obtained fluorescence images, selecting four indicators for analysis: average area, average branch length, average fluorescence intensity, and average network number. Figure 10 and Figure 12 The results showed that α-synuclein oligomer 2 (O2) was the most neurotoxic.

[0079] 4.3.3) Specific implementation method for reactive oxygen species level detection:

[0080] DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it can be hydrolyzed into DCFH by esterases. DCFH cannot cross the cell membrane, so it can be easily loaded into the cell. When reactive oxygen species (ROS) are present in the cell, the non-fluorescent DCFH can be oxidized to DCF, which has green fluorescence. The level of ROS in the cell can be determined by detecting the fluorescence of DCF. The DCFH-DA probe is used to detect the level of intracellular ROS, and the fluorescence spectrum is obtained from the results. Figure 13 It can be seen that the reactive oxygen species content in cells treated with oligomer 1 (O1) and oligomer 2 (O2) increased significantly. Morphological quantitative analysis was also performed on the obtained fluorescence images, selecting four indicators for analysis: average area, average branch length, average fluorescence intensity, and average network number. Figure 14 The results also showed that oligomer 2 was the most neurotoxic. This further validates the usability of the α-synuclein aggregates prepared by the method of this invention.

[0081] Furthermore, this invention successfully prepared α-synuclein aggregates of various sizes by adjusting process parameters within the aforementioned process range and sampling and purifying at different incubation time points (e.g., 4h, 10h, 16h, 25h, 36h, and 47h depending on the required size). The entire process is simple and efficient, and the target product is stable and uniform. The prepared α-synuclein aggregates can not only be used for pathological research of Parkinson's disease and the establishment of in vitro disease models, but also for pathological research and model establishment of other α-synuclein diseases such as Lewy body dementia and multiple system atrophy.

Claims

1. A rapid method for preparing multi-sized α-synuclein aggregates, characterized in that, Includes the following steps: 1) Dissolve the isolated and purified α-synuclein monomers in 4 mL of water according to the concentration standard of 1 mg / mL. o C was added to PBS buffer solution and mixed well to obtain a monomeric protein solution; 2) Place the monomeric protein solution obtained in step 1) into a centrifuge tube and incubate at 37°C. o The samples were incubated at 500 rpm under shaking conditions, and samples were taken after 6 h, 15 h, 24 h and 48 h, with each sample being no less than 200 μL. 3) Add the samples obtained in step 2) to 100 kDa ultrafiltration centrifuge tubes and incubate at 4°C. o C. Centrifuge at 15300 g for 5 min to separate the monomers from the aggregates; 4) Add 100 μL of PBS buffer solution to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into the centrifuge tube it is paired with. Incubate at 4°C. o C. Centrifuge at 450 g for 4 min, collect protein aggregates on the inner membrane of each ultrafilter, obtain protein aggregates of different sizes, and store at 4... o C is available for later use.

2. Multi-sized α-synuclein aggregates, characterized in that, The sample was prepared using the rapid preparation method described in claim 1 at different incubation times.

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