A method for amyloid protein aggregate enrichment and dissolution based on ionic liquid pairs

Abstract

The application discloses a method for enriching and dissolving amyloid protein aggregates based on an ionic liquid pair. The method is aimed at the problem of poor solubility caused by the special cross beta-sheet structure of amyloid protein aggregates. The method uses the characteristics of multiple anion and cation types and adjustable structure to select an "imidazolium ionic liquid-tetramethyl guanidine ionic liquid pair" for the enrichment and dissolution of amyloid protein aggregates. The method has simple and robust steps, high recovery rate and good mass spectrum compatibility, and can be used for high selectivity and high sensitivity analysis of amyloid protein aggregates.

Description

Technical Field

[0001] This invention relates to a method for enriching and dissolving amyloid protein aggregates based on ionic liquid extraction, and establishes an amyloid protein aggregate enrichment and analysis platform based on novel ionic liquid pairs, belonging to the field of protein sample preparation technology. Background Technology

[0002] Amyloid deposition is a key pathological marker of neurodegenerative diseases (Cell, 2023, 186(4), 693-714; Nat. Rev. Neurol., 2020, 16(4), 199-212). A class of amyloid proteins, represented by Tau, Aβ, and α-synuclein, tend to aggregate with other proteins to form amyloid fibrils and gradually deposit. These amyloid aggregates not only impair cellular function but are also associated with pathological processes such as neuroinflammation and oxidative stress, thereby accelerating disease progression (Nat. Neurosci., 2024, 27(10), 1880-1891; Cell Reports, 2022, 40(12), 111401). Therefore, a deeper understanding of the composition and formation mechanism of amyloid aggregates can provide new insights for the early diagnosis and effective treatment of neurodegenerative diseases.

[0003] Currently, mass spectrometry (MS)-based methods have become powerful tools for amyloid aggregate proteomics analysis. Among them, multi-step centrifugation-mass spectrometry analysis is the most widely used method in amyloid aggregate proteomics research. For example, Upadhyay et al. developed a density gradient-ultracentrifugation tandem ultrasound extraction method to analyze amyloid protein aggregates and newly identified 20 Aβ42 interacting proteins that regulate amyloid protein formation (Mol. Neurodegener., 2023, 18(1), 61). Similarly, Zaman et al. used differential centrifugation tandem TMT labeling mass spectrometry to identify 84 detergent-insoluble amyloid aggregate proteins from postmortem brain tissue of Alzheimer's disease (AD) patients (Mol. Cell Proteomics, 2023, 22(8), 100608). However, although the dense structure of amyloid protein aggregates leads to their centrifugal force and water solubility that differ from other proteins, the complexity of cellular components limits the selectivity and recovery efficiency of amyloid protein aggregates separated by these methods. Meanwhile, laser microdissection enrichment-mass spectrometry is also a commonly used method for proteomic analysis of amyloid aggregates. Drummond et al. combined immunohistochemistry with laser microdissection-mass spectrometry to identify 542 neurofibrillary tangle-related proteins in the hippocampus of patients with advanced Alzheimer's disease (Brain, 2020, 143(9), 2803-2817). However, limited by imaging resolution, these methods capture all proteins inside and around amyloid aggregates, which may include spatially adjacent but functionally unrelated proteins, thus reducing the method's ability to accurately identify key proteins directly involved in amyloid aggregation. In addition, proximity labeling-mass spectrometry has also been used for compositional analysis of protein aggregates. These methods utilize enzymes with proximity labeling functions (BioID, APEX2, etc.) or small molecule probes to specifically target key molecules in amyloid aggregates, thereby achieving selective labeling and enrichment of these aggregates (Nat. Neurosci, 2018, 21(2), 228-239; Mol. Neurodegener., 2025, 20(1), 2). For example, Chou et al. used BioID to map the interactionome of TDP-43 aggregates, revealing that TDP-43-mediated nuclear-cytoplasmic transport defects are a key pathological mechanism of ALS / FTD28 (Nat. Neurosci, 2018, 21(2), 228-239). However, these methods often target not only amyloid protein aggregates but also non-amyloid aggregates, thus reducing the accuracy of amyloid aggregate composition identification. Therefore, highly selective and accurate amyloid aggregate proteomic analysis methods are currently lacking.

[0004] However, due to the low abundance and water insolubility of amyloid aggregates, their enrichment and dissolution remain significant challenges, which are crucial steps in the comprehensive identification of the amyloid aggregate proteome. Therefore, there is an urgent need to develop extraction and enrichment methods specifically targeting the unique structural features of amyloid aggregates to deepen our understanding of their composition, aggregation mechanisms, and related pathologies.

[0005] Ionic liquids are a class of molten salts composed of cations and anions. The cations and anions that make up ionic liquids are diverse, exhibiting rich interactions with proteins. This endows ionic liquids with tunable structural properties and multifunctionality, allowing them to be designed to interact with proteins in different ways (Trac-Trend Anal. Chem., 2018, 108, 239-246). Currently, ionic liquids have shown advantages in the extraction of soluble proteins and the stabilization of protein complex structures (Mol. Cell Proteomics, 2020, 19(10): 1724-1737; Anal. Chem., 2022, 94(2), 758-767). Therefore, based on the rich interactions and tunable structural properties between ionic liquids and proteins, this invention establishes a novel method for the enrichment and dissolution analysis of amyloid protein aggregates based on a novel ionic liquid pair. The two ionic liquids have orthogonal interactions with the protein, thereby achieving highly selective and accurate identification of amyloid protein aggregates. Summary of the Invention

[0006] To overcome the challenges of enrichment and dissolution posed by the low abundance and water insolubility of amyloid protein aggregates and to improve the identification coverage of amyloid aggregate proteomes, this invention establishes a method for enrichment and dissolution of amyloid protein aggregates based on novel ionic liquid pairs.

[0007] A method for enriching and dissolving amyloid protein aggregates based on ionic liquid pairs utilizes a pair of orthogonally interacting ionic liquids. The method involves first using an imidazole-based ionic liquid to dissolve soluble proteins and non-amyloid aggregates, followed by using a tetramethylguanidine-based ionic liquid to specifically dissolve the amyloid protein aggregates. Specifically, the method includes:

[0008] (1) Tissue or cell samples are first broken up, and the supernatant solution after centrifugation is labeled as the initial fraction. The precipitate is the protein sample containing amyloid protein aggregates after the breakup.

[0009] (2) Prepare a methylimidazolium ionic liquid solution (extraction solution A) using a weak alkaline buffer solution and add auxiliary additives. Add the methylimidazolium ionic liquid solution (extraction solution A) to the precipitated protein sample after step (1) to dissolve soluble proteins and non-amyloid aggregates. After ultrasonic extraction of these proteins, centrifuge the sample and collect the supernatant, labeling it as fraction A. The precipitated protein is the enriched amyloid protein aggregate.

[0010] (3) Prepare tetramethylguanidine cationic liquid (extraction solution B) using a weak alkaline buffer and add auxiliary additives. Add tetramethylguanidine cationic liquid (extraction solution B) to the precipitated protein obtained in step (2) to dissolve the amyloid protein aggregates. After ultrasonic extraction of the protein, centrifuge the sample and collect the supernatant. The supernatant is labeled as fraction B, which is the dissolved amyloid protein aggregate.

[0011] Methylimidazole ionic liquids dissolve proteins and non-amyloid aggregates through hydrophobic interactions, while tetramethylguanidine cationic liquids dissolve amyloid protein aggregates through hydrogen bonding.

[0012] After enriching and dissolving amyloid protein aggregates, qualitative and quantitative analysis of the amyloid protein samples is required. The specific steps are as follows: the labeled fractionated protein solutions (supernatant) in steps (1)-(3) are added to the filter membrane of the protein centrifugation ultrafiltration device to perform protein denaturation, reduction alkylation and protein enzymatic digestion. Then, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is performed to perform qualitative and quantitative analysis of the amyloid protein in the extract.

[0013] The ionic liquid used in extraction solution A is one or more of methylimidazolium cations containing 8-12 carbon atoms in an alkyl chain, with the following structural formula:

[0014]

[0015] The anion extracted from solution A is chloride ion, and the mass-volume ratio of ionic liquid to weakly alkaline buffer is 1%-20% (preferably 5%-16%, more preferably 8%-13%).

[0016] The ionic liquid used to extract solution B has a tetramethylguanidine cation, with the following structural formula:

[0017]

[0018] The anions in the extraction solution B are one or both of tetrafluoroborate anions and chloride ions, and the concentration of the ionic liquid is 1-20 mol / L (preferably 5%-16%, more preferably 8%-13%).

[0019] The structural formula of the cation of the ionic liquid used to extract solution A is as follows:

[0020]

[0021] The weakly alkaline buffer solution is composed of tris(hydroxymethyl)aminomethane buffer solution or phosphate buffer solution, with a pH of 7.2-8.0; the auxiliary additive is one or both of protease inhibitors and phosphatase inhibitors; the volume concentration of the protease inhibitor in the weakly alkaline buffer solution is 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%), and the volume concentration of the phosphatase inhibitor in the weakly alkaline buffer solution is 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%).

[0022] The sample used in step (1) is an animal tissue or cell with an amyloid aggregation phenotype.

[0023] The tissue was derived from the brain tissue of a mouse model of neurodegenerative disease, and the cells were derived from cells that highly expressed amyloid protein aggregates.

[0024] The tissue or cell samples in step (1) need to be homogenized. First, add homogenization buffer, which is a phosphate buffer solution (pH = 7.2-8.0) containing 1%-5% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%) of one or more of NP-40 (octylphenyl-polyethylene glycol), Triton X-100 and Tween 20, with a volume concentration of 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%) of protease inhibitor and 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%) of phosphatase inhibitor. At a temperature of -30℃ to -50℃, homogenize the tissue or cells 8-15 times using a cryogenic homogenizer with a bead diameter of 1-4 mm and a homogenization speed of 4-6 m / s.

[0025] The ultrasound conditions for steps (2) and (3) are to use 60-150W power for 3-30 minutes on ice;

[0026] The sample centrifugation conditions in steps (1)-(3) are 16000g-30000×g, 0-4℃, and centrifugation time is 20min-1h.

[0027] The specific steps of the qualitative and quantitative analysis are as follows:

[0028] First, denature the protein solutions labeled in steps (1)-(3) using a protein denaturing reagent; then transfer the solution to a protein-retaining filter membrane, centrifuge to remove the solution on the membrane, at which point the protein is retained on the filter membrane surface; wash the filter membrane surface with ammonium bicarbonate solution to remove excess protein denaturing reagent, centrifuge to remove the solution; then use an alkylating reagent to reduce and alkylate the precipitated protein on the filter membrane surface, centrifuge to remove the alkylating reagent solution on the filter membrane after the reaction; wash the filter membrane 2-5 times with urea-NH4HCO3 solution, then wash the filter membrane 2-5 times with NH4HCO3 solution; then add NH4HCO3 and protein digestion reagent to perform protein digestion; after the protein digestion reaction, collect the obtained peptide solution and perform LC-MS / MS analysis;

[0029] The protein ultrafiltration membrane used for qualitative and quantitative analysis had a protein size cutoff of 3-30 kDa. The protein denaturing reagent was dithiothreitol (DTT) at a concentration of 10-100 mM, with denaturation conditions of 37-56℃ for 1-2 hours. The alkylation reagent was iodoacetamide (IAA) at a concentration of 20-50 mM, with alkylation conditions of 30 minutes in the dark. The protein enzymatic digestion reagent was Trypsin, used at an enzyme:protein (m / m) ratio of 1:25-1:50, with digestion conditions of 37℃ for 12-16 hours. The centrifugation conditions for the ultrafiltration device in the qualitative and quantitative analysis steps were 14000-16000 g, 15-25℃, for 20 minutes to 1 hour.

[0030] The specific technical solution adopted in this invention is as follows:

[0031] (1) Enrichment of amyloid aggregate proteins:

[0032] For tissue samples, add 0.1-10 ml of NP-40 homogenization buffer (0.5%-5% v / v concentration, containing 0.5%-3% protease and phosphatase inhibitors) to 10 mg-1 g of tissue sample for homogenization. The homogenization environment temperature is -30 to -50℃, the homogenization speed is 4-6 m / s, the number of homogenization cycles is 8-15, and the diameter of the homogenate beads is 1-4 mm. Centrifuge the homogenate at 16000-30000×g for 0.5-1 h, collect the supernatant, and label it as fraction A. Add 0.1-10 ml of C12ImCl ionic liquid-phosphate buffer to the precipitate, with the mass (g) of the ionic liquid to the volume (mL) of the weakly alkaline buffer being 5%-30%. Use sonication to extract soluble proteins and non-amyloid aggregates from the precipitate under the following conditions: 60-150 W, 3-30 min, 0-4℃. The samples were then centrifuged at 16000-30000×g for 20 min-1 h, and the supernatant was collected and labeled as C12ImCl fraction. At this point, amyloid protein aggregates were enriched in the precipitate.

[0033] For cell samples, 0.2-5 ml of C12ImCl ionic liquid-phosphate buffer was added to cells at 1e7-1e9 mmol / L. The mass (g) of the ionic liquid to the volume (mL) of the weakly alkaline buffer was 5%-30%, and the buffer contained 0.5-3% (v / v) protease and phosphatase inhibitors. Soluble proteins and non-amyloid aggregates were extracted using sonication-assisted extraction at 60-150 W for 3-30 min at 0-4 °C. The samples were then centrifuged at 16000-30000 × g for 20 min-1 h, and the supernatant was collected and labeled as the C12ImCl fraction. At this point, amyloid protein aggregates were enriched in the precipitate.

[0034] (2) Dissolution of amyloid aggregates:

[0035] For cell or tissue samples, 0.2-5 ml of TMGBF4 ionic liquid-phosphate buffer (1-20 mol / L) containing 0.5-3% (v / v) protease and phosphatase inhibitors is added to the precipitate after C12 ImCl extraction. The amyloid protein aggregates are then dissolved using sonication at 60-150 W for 3-30 min at 0-4 °C. The sample is then centrifuged at 16000-30000 × g for 20 min-1 h, and the supernatant is collected and labeled as the TMGBF4 fraction. This fraction is enriched with amyloid protein aggregates.

[0036] (3) Preparation and large-scale identification of amyloid aggregation proteomics samples

[0037] The three fractions (fraction A, fraction C12ImCl, and fraction TMBGF4) described in (1)-(2) were processed using a membrane-assisted proteomics sample preparation method, with the membrane used having a protein cutoff size of 10 kDa. The prepared peptides were then analyzed by LC-MS / MS.

[0038] (4) Data Analysis

[0039] Mass spectrometry data were retrieved using Plind software, and the peak intensities of identified proteins were analyzed using pQuant software. Protein information identified by different fractions was compared. Among proteins identified by both the TMBF4 and C12ImCl fractions, proteins with peak intensities in the TMBF4 fraction greater than or equal to those in the C12ImCl fraction were defined as TMBF4 enriched fraction proteins. These proteins are constituent proteins of amyloid aggregates and are expected to provide fundamental data for a deeper understanding of the pathological mechanisms of amyloid aggregates in neurodegenerative diseases.

[0040] The present invention has the following advantages:

[0041] 1. Method: A pair of ionic liquids that are complementary to the protein interaction were used to achieve highly selective and high-recovery enrichment and dissolution of amyloid protein aggregates.

[0042] 2. The hydrogen bond interaction between TMGBF4 and proteins enables it to specifically recognize and disrupt the typical cross-beta-sheet structure of amyloid aggregates, thereby achieving specific dissolution of amyloid aggregate proteins.

[0043] 3. The method is robust and highly operable. Both ionic liquids are compatible with common proteomics sample preparation methods, and when combined with mass spectrometry analysis, they enable large-scale identification of amyloid aggregates. Attached Figure Description

[0044] Figure 1 The structural formula of the ionic liquid, wherein: Figure 1 (A) is a methylimidazolium-based ionic liquid solution. (General formula for ionic liquids) Figure 1 (B) is a TMGBF4 ionic liquid. Figure 1 (C) is a C12ImCl ionic liquid.

[0045] Figure 2 A simplified flowchart of the method described in this invention applied to tissue samples. Detailed Implementation

[0046] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0047] Example 1

[0048] The technical solution adopted in this invention is as follows:

[0049] (1) Enrichment of amyloid protein aggregates

[0050] 500 μL of hippocampal tissue from 12-month-old female 3xTg mice (3xTg is a transgenic mouse model of Alzheimer's disease (AD), containing three mutations: APP Swedish, MAPT P301L, and PSEN1M146V, exhibiting typical pathological features of Alzheimer's disease, purchased from Shulaibao Biotechnology Co., Ltd.) was added to PBS (pH=7.4) containing 1% (v / v) of a protease inhibitor (purchased from Sigma, P8215, the same below) and 1% (v / v) of a phosphatase inhibitor (purchased from Sigma, P5726, the same below). The sample was then homogenized. The homogenization conditions were as follows: tissue homogenization was performed 10 times at a temperature of -40℃ using a cryogenic grinder with a grinding bead diameter of 2mm and a grinding speed of 4.5m / s.

[0051] The homogenized sample was centrifuged at 20000×g for 1 h at 4℃. The supernatant was collected as the NP-40 fraction (initial fraction). 500 μL of C12ImCl ionic liquid-phosphate buffer (the C12ImCl ionic liquid is 1-dodecyl-3-methylimidazolium chloride, purchased from Lanzhou Greenchem ILs, CP0161, with the structural formula shown below) was added to the precipitate. Figure 1 B), wherein the mass (g) of C12ImCl ionic liquid is 10% of the volume (mL) of phosphate buffer (pH = 7.4), and the solution contains 1% (v / v) protease inhibitor and 1% (v / v) phosphatase inhibitor. Soluble proteins and non-amyloid aggregates in the precipitate were extracted using ultrasound-assisted extraction at 100 W for 20 min at 4 °C. The sample was then centrifuged at 30000 × g for 0.5 h, and the supernatant was collected and labeled as the C12ImCl fraction.

[0052] (2) Dissolution of amyloid protein aggregates

[0053] The precipitate after C12ImCl fractionation was added to 500 μL of TMGBF4 ionic liquid-phosphate buffer (pH = 7.4) (the TMGBF4 ionic liquid solute was tetramethylguanidine tetrafluoroborate, purchased from Lanzhou Yulu Fine Chemical Co., Ltd., and its structural formula is shown below). Figure 1 C) The concentration was 10 mol / L, containing 1% (v / v) protease inhibitor and 1% (v / v) phosphatase inhibitor. Amyloid protein aggregates were dissolved using sonication at 100 W for 20 min at 4 °C. The sample was then centrifuged at 30000 × g for 0.5 h, and the supernatant was collected and labeled TMGBF4 fraction.

[0054] (3) Preparation and large-scale identification of amyloid aggregation proteomics samples

[0055] NP-40 fraction (initial fraction), C12ImCl fraction, and TMGBF4 fraction were processed using a membrane-assisted proteomics sample preparation method. 150 μg of protein solution was taken from each fraction and dithiothreitol (DTT) was added to a final concentration of 100 mM. The mixture was reacted at 56 °C for 1 h to denature the proteins. The protein solution was then transferred to a 10 kDa protein-retaining membrane (purchased from Sartorius AG), centrifuged at 14000 × g for 30 min, and the solution on the membrane was removed. At this point, the protein was retained on the membrane surface. Then, 200 μL of 50 mM ammonium bicarbonate (NH4HCO3) solution was added to the membrane surface to wash away any remaining DTT solution. The membrane was centrifuged at 14000 × g for 30 min to remove the washing solution. Then, 200 μL of iodoacetamide (IAA) with a final concentration of 20 mM was added to the filter membrane surface to alkylate the proteins on the membrane surface. The reaction was carried out at room temperature in the dark for 30 min, followed by centrifugation at 14000 × g for 30 min to remove the IAA solution. The filter membrane was washed twice with 200 μL of 8 M urea-50 mM NH4HCO3 solution, and then three times with 200 μL of 50 mM NH4HCO3 solution, each time centrifuged at 14000 × g for 30 min. Then, 50 μL of 10 mM NH4HCO3 solution was added, and trypsin was added at a protein:enzyme ratio of 25:1. The membrane was incubated at 37 °C for 14 h. The target proteins remained on the filter membrane surface throughout the above steps. The membrane solution was then centrifuged (14000 × g, 25 °C, 30 min), and the obtained peptides were analyzed by LC-MS / MS.

[0056] (4) Data Analysis

[0057] Mass spectrometry data were retrieved using Plind software, and the peak intensities of the identified proteins were analyzed using pQuant software. Protein information identified by different fractions was compared: 5979 proteins were identified in the NP40 fraction, 6061 proteins in the C12ImCl fraction, and 5050 proteins in the TMBDF4 fraction. Among the proteins identified by both the NP40 and C12ImCl fractions, proteins with peak intensities at least twice that of the NP40 fraction in the C12ImCl fraction were defined as C12ImCl-enriched proteins, totaling 1102 proteins. Similarly, among the proteins identified by both the TMBDF4 and C12ImCl fractions, proteins with peak intensities at least twice that of the C12ImCl fraction in the TMBDF4 fraction were defined as TMBDF4-enriched proteins, totaling 1185 proteins. Comparing proteins enriched by C12ImCl and those enriched by TMGBF4, it was found that the TMGBF4-enriched proteins had a higher average phase separation score and a higher proportion of disordered regions (as shown in Appendix Table 1), indicating that the TMGBF4-enriched proteins have a higher tendency to aggregate into amyloid aggregates. Compared with previously reported amyloid aggregate constituent proteins (average abundance 888), 922 new amyloid aggregation-related proteins were identified in the TMGBF4-enriched proteins. These proteins had lower abundance (average abundance 95.8), indicating that the method can more sensitively enrich amyloid aggregate constituent proteins with lower abundance.

[0058] Example 2

[0059] The steps were the same as in Example 1, except that the ionic liquid used in step (2) was TMGCl (tetramethylguanidine chloride, purchased from Lanzhou Yulu Fine Chemical Co., Ltd.). Data analysis results showed that C12ImCl enriched 1050 proteins, while TMGCl enriched 1123 proteins. The average phase separation score and the proportion of disordered regions of the proteins enriched by TMGCl were higher than those enriched by C12ImCl (phase separation score: 0.63 vs 0.52; proportion of disordered regions: 40.7% vs 33.2%), indicating that the cationic portion of the ionic liquid was the main dissolving agent for amyloid protein aggregates.

[0060] Example 3

[0061] The steps were the same as in Example 1, except that the tissue sample used was cortical brain tissue from 12-month-old female 3xTg mice (3xTg is an AD model transgenic mouse containing three mutations: APP Swedish, MAPT P301L, and PSEN1 M146V, exhibiting typical AD pathological characteristics; purchased from Shulaibao Biotechnology Co., Ltd.). Data analysis results showed that C12ImCl enriched 1150 proteins, while TMGBF4 enriched 970 proteins. The average phase separation score and the percentage of disordered regions of the TMGBF4-enriched proteins were higher than those of the C12ImCl-enriched proteins (phase separation score: 0.60 vs 0.43; percentage of disordered regions: 39.2% vs 30.1%), indicating that the method is applicable to tissue regions with amyloid aggregation phenotypes in neurodegenerative disease model mice.

[0062] Example 4

[0063] The steps were the same as in Example 1, except that the tissue sample used was hippocampal tissue from 6-month-old female 5xFAD mice (5xFAD mice are transgenic AD model mice that simultaneously express five familial AD mutant genes and exhibit typical pathological features of Alzheimer's disease; purchased from Shulaibao Biotechnology Co., Ltd.). Data analysis showed that C12ImCl enriched 876 proteins, while TMGBF4 enriched 1127 proteins. The average phase separation score and the percentage of disordered regions of the TMGBF4-enriched proteins were higher than those of the C12ImCl-enriched proteins (phase separation score: 0.66 vs 0.53; percentage of disordered regions: 40.5% vs 32.4%), indicating that the method is applicable to tissue regions with amyloid aggregation phenotypes in neurodegenerative disease model mice.

[0064] Example 5

[0065] The steps were the same as in Example 1, except that the sample used was a tissue sample from the cerebral cortex of 12-month-old female A53T mice (A53T mice are PD model transgenic mice carrying the A53T mutant human α-synuclein gene, exhibiting typical pathological features of Parkinson's disease). Data analysis showed that C12ImCl enriched 648 proteins, while TMGBF4 enriched 739 proteins. The average phase separation score and the percentage of disordered regions of the TMGBF4-enriched proteins were higher than those of the C12ImCl-enriched proteins (phase separation score: 0.62 vs 0.51; percentage of disordered regions: 38.4% vs 29.1%), indicating that the method is applicable to tissue regions with amyloid aggregation phenotypes in neurodegenerative disease model mice.

[0066] Example 6

[0067] The steps are the same as in Example 1, except that the sample is HEK293T cells highly expressing the Tau p301L gene. The NP-40-phosphate buffer homogenization steps in Example 1 are omitted when processing the cell samples. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 8 250 μL of C12ImCl ionic liquid-phosphate buffer (pH=7.4) was added to each cell. The mass (g) to volume (mL) ratio of the ionic liquid to the phosphate buffer (pH=7.4) was 10%, and the buffer contained 1% (v / v) protease inhibitor and 1% (v / v) phosphatase inhibitor. Soluble proteins and non-amyloid aggregates were extracted using sonication at 60 W for 10 min at 4 °C. The samples were then centrifuged at 20000 × g for 0.5 h, and the supernatant was collected and labeled as the C12ImCl fraction. 250 μL of TMGBF4 ionic liquid-phosphate buffer (pH=7.4), 10 mol / L, containing 1% (v / v) protease inhibitor and 1% (v / v) phosphatase inhibitor, was added to the precipitate. Amyloid protein aggregates were dissolved using sonication at 60 W for 10 min at 4 °C. The sample was then centrifuged at 20000×g for 0.5h, and the supernatant was collected and labeled as the TMBF4 fraction. The remaining steps were the same as in Example 1 (3)-(4). Data analysis results showed that there were 1056 proteins in the C12ImCl fraction and 645 proteins in the TMBF4 fraction. 248 proteins were enriched by TMBF4, including typical amyloid proteins such as Tau and Aβ, indicating that the method is also suitable for enriching intracellular amyloid aggregation proteins.

[0068] Example 7

[0069] The steps are the same as in Example 1, except that the sample is HT22 cells highly expressing the Tau p301L gene. The NP-40-phosphate buffer homogenization steps in Example 1 are omitted when processing the cell samples. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 8250 μL of C12ImCl ionic liquid-phosphate buffer (pH=7.4) was added to each cell. The mass (g) to volume (mL) ratio of the ionic liquid to the phosphate buffer (pH=7.4) was 10%, and the buffer contained 1% (v / v) protease inhibitor and 1% (v / v) phosphatase inhibitor. Soluble proteins and non-amyloid aggregates were extracted using sonication at 60 W for 10 min at 4 °C. The samples were then centrifuged at 20000 × g for 0.5 h, and the supernatant was collected and labeled as the C12ImCl fraction. 250 μL of TMGBF4 ionic liquid-phosphate buffer (pH=7.4), 10 mol / L, containing 1% (v / v) protease inhibitor and 1% (v / v) phosphatase inhibitor, was added to the precipitate. Amyloid protein aggregates were dissolved using sonication at 60 W for 10 min at 4 °C. The sample was then centrifuged at 20000×g for 0.5h, and the supernatant was collected and labeled as the TMGBF4 fraction. The remaining steps were the same as in (3)-(4) of Example 1. Data analysis results showed that there were 889 proteins in the C12ImCl fraction and 473 proteins in the TMGBF4 fraction. TMGBF4 enriched 104 proteins, including typical amyloid proteins such as Tau and Aβ, indicating that the method is also suitable for enriching intracellular amyloid aggregation proteins.

[0070] Comparative Example 1

[0071] The method and steps are derived from the literature (Phosphorylated tau interactome in the human Alzheimer's disease brain. Brain, 2020, 143(9), 2803-2817), and are summarized as follows:

[0072] Immunohistochemical staining (AT8 anti-phosphorylated tau antibody) was performed on post-mortem brain tissue (hippocampus and entorhinal cortex) from Alzheimer's patients. Then, regions showing AT8-positive staining and typical NFT morphology were selected, and neurofibrillary tangles were extracted using laser capture microdissection (LCM). Approximately 4000 NFTs (1.5 mm²) were collected from each sample. Proteins were extracted by treatment in formic acid solution. The protein solution extracted from the literature was centrifuged (14000 × g, 25°C, 30 min) and then digested with trypsin (trypsin added at a protein:enzyme (m / m) ratio of 25:1, incubated at 37°C for 14 h). Mass spectrometry analysis was performed using LC-MS / MS. Data analysis showed that a total of 542 neurofibrillary tangle proteins were identified. Among them, 103 proteins were also identified in the TMBGF4 enrichment fraction in Example 1. Furthermore, the average protein abundance of Comparative Example 1 was 888, spanning seven orders of magnitude. The average abundance of TMBGF4-enriched proteins in Example 1 was 170, spanning eight orders of magnitude. These results demonstrate that the method exhibits higher sensitivity for the enrichment of amyloid proteins.

[0073] Table 1. Comparison of average phase separation scores and average proportions of disordered regions for proteins enriched by C12ImCl and TMGBF4.

[0074]

[0075]

Claims

1. A method for enriching and dissolving amyloid protein aggregates based on ionic liquid pairs, characterized in that: The enrichment and dissolution of amyloid protein aggregates is performed using a pair of orthogonally interacting ionic liquids. First, imidazole ionic liquids are used to dissolve soluble proteins and non-amyloid aggregates, and then tetramethylguanidine ionic liquids are used to specifically dissolve the amyloid protein aggregates. Specifically, this involves: (1) Tissue or cell samples are first broken up, and the supernatant solution after centrifugation is labeled as the initial fraction. The precipitate is the protein sample containing amyloid protein aggregates after the breakup. (2) Prepare a methylimidazolium ionic liquid solution (extraction solution A) using a weak alkaline buffer solution and add auxiliary additives. Add the methylimidazolium ionic liquid solution (extraction solution A) to the precipitated protein sample after step (1) to dissolve soluble proteins and non-amyloid aggregates. After ultrasonic extraction of these proteins, centrifuge the sample and collect the supernatant, labeling it as fraction A. The precipitated protein is the enriched amyloid protein aggregate. (3) Prepare tetramethylguanidine cationic liquid (extraction solution B) using a weak alkaline buffer and add auxiliary additives. Add tetramethylguanidine cationic liquid (extraction solution B) to the precipitated protein obtained in step (2) to dissolve the amyloid protein aggregates. After ultrasonic extraction of the protein, centrifuge the sample and collect the supernatant. The supernatant is labeled as fraction B, which is the dissolved amyloid protein aggregate.

2. The method according to claim 1, characterized in that: Methylimidazole ionic liquids dissolve proteins and non-amyloid aggregates through hydrophobic interactions, while tetramethylguanidine cationic liquids dissolve amyloid protein aggregates through hydrogen bonding.

3. The method according to claim 1, characterized in that, After enriching and dissolving amyloid protein aggregates, qualitative and quantitative analysis of the amyloid protein samples is required. The specific steps are as follows: the labeled fractionated protein solutions (supernatant) in steps (1)-(3) are added to the filter membrane of the protein centrifugation ultrafiltration device to perform protein denaturation, reduction alkylation and protein enzymatic digestion. Then, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is performed to perform qualitative and quantitative analysis of the amyloid protein in the extract.

4. The method according to claim 1, characterized in that: The ionic liquid used in extraction solution A is one or more of methylimidazolium cations containing 8-12 carbon atoms in an alkyl chain, with the following structural formula: The anion extracted from solution A is chloride ion, and the mass-volume ratio of ionic liquid to weakly alkaline buffer is 1%-20% (preferably 5%-16%, more preferably 8%-13%). The ionic liquid used to extract solution B has a tetramethylguanidine cation, with the following structural formula: The anions in the extraction solution B are one or both of tetrafluoroborate anions and chloride ions, and the concentration of the ionic liquid is 1-20 mol / L (preferably 5%-16%, more preferably 8%-13%).

5. The method according to claim 1 or 3, characterized in that: The structural formula of the cation of the ionic liquid used to extract solution A is as follows:

6. The method according to claim 1, characterized in that, The weakly alkaline buffer solution is composed of tris(hydroxymethyl)aminomethane buffer solution or phosphate buffer solution, with a pH of 7.2-8.0; the auxiliary additive is one or both of protease inhibitors and phosphatase inhibitors; the volume concentration of the protease inhibitor in the weakly alkaline buffer solution is 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%), and the volume concentration of the phosphatase inhibitor in the weakly alkaline buffer solution is 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%).

7. The method according to claim 1, characterized in that, The sample used in step (1) is an animal tissue or cell with an amyloid aggregation phenotype.

8. The method according to claim 7, characterized in that, The tissue was derived from the brain tissue of a mouse model of neurodegenerative disease, and the cells were derived from cells that highly expressed amyloid protein aggregates.

9. The method according to claim 1 or 6, characterized in that, The tissue or cell samples in step (1) need to be homogenized. First, add homogenization buffer, which is a phosphate buffer solution (pH = 7.2-8.0) containing 1%-5% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%) of one or more of NP-40 (octylphenyl-polyethylene glycol), Triton X-100 and Tween 20, with a volume concentration of 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%) of protease inhibitor and 0.5%-3% (preferably 0.6%-2.5%, more preferably 0.8%-1.5%) of phosphatase inhibitor. At a temperature of -30℃ to -50℃, homogenize the tissue or cells 8-15 times using a cryogenic homogenizer with a bead diameter of 1-4 mm and a homogenization speed of 4-6 m / s. The ultrasound conditions for steps (2) and (3) are to use 60-150W power for 3-30 minutes on ice; The sample centrifugation conditions in steps (1)-(3) are 16000g-30000×g, 0-4℃, and centrifugation time is 20min-1h.

10. The method according to claim 1 or 3, characterized in that, The specific steps of the qualitative and quantitative analysis are as follows: First, denature the protein solution labeled fractions in steps (1)-(3) using a protein denaturing reagent; then transfer the solution to a protein-retaining filter membrane, centrifuge to remove the solution on the membrane, at which point the protein is retained on the surface of the filter membrane. Use ammonium bicarbonate solution to clean the surface of the filter membrane of any remaining protein denaturing reagent, and then centrifuge to remove the solution. Then, the precipitated proteins on the filter membrane surface were reduced and alkylated using an alkylating agent. After the reaction, the alkylating agent solution on the filter membrane was removed by centrifugation. The filter membrane was washed 2-5 times with urea-NH4HCO3 solution, and then washed 2-5 times with NH4HCO3 solution. Then, NH4HCO3 and protein digestion reagent were added to perform protein digestion. After the protein digestion reaction, the obtained peptide solution was collected and analyzed by LC-MS / MS. The protein ultrafiltration membrane used for qualitative and quantitative analysis had a protein size cutoff of 3-30 kDa. The protein denaturing reagent was dithiothreitol (DTT) at a concentration of 10-100 mM, with denaturation conditions of 37-56℃ for 1-2 hours. The alkylation reagent was iodoacetamide (IAA) at a concentration of 20-50 mM, with alkylation conditions of 30 minutes in the dark. The protein enzymatic digestion reagent was Trypsin, used at an enzyme:protein (m / m) ratio of 1:25-1:50, with digestion conditions of 37℃ for 12-16 hours. The centrifugation conditions for the ultrafiltration device in the qualitative and quantitative analysis steps were 14000-16000 g, 15-25℃, for 20 minutes to 1 hour.

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