Enrichment and identification of tau protein variants in neurodegenerative diseases
By using magnetic beads functionalized with tau recognition peptides for specific enrichment and a top-down/middle-down proteomics strategy, the challenge of identifying tau protein variants has been solved, achieving highly specific enrichment and accurate identification of tau protein variants, applicable to cell and tissue samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN INSTITUTE OF CHEMICAL PHYSICS CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2024-11-28
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies suffer from poor specificity for enriching tau proteins and their large molecular weight makes accurate identification difficult, leading to challenges in analyzing tau protein variants.
By employing magnetic spheres specifically enriched with tau recognition peptides and combining top-down and middle-down proteomics strategies, tau proteins were enriched through the synthesis of functionalized magnetic spheres and identified using liquid chromatography-mass spectrometry (LC-MS), enabling precise analysis of tau protein variants.
It achieves highly specific enrichment and accurate identification of tau protein variants, enabling comprehensive study of post-translational modifications, sequence variations, and truncation information, and is applicable to cell and tissue samples.
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Figure CN122108723A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for enriching and identifying tau protein variants in neurodegenerative diseases, belonging to the field of bioanalytical technology. Background Technology
[0002] Tau protein is a focal point of research in neurodegenerative diseases. Understanding how it produces toxicity and leads to cognitive decline holds promise for new therapies for these diseases. Numerous tau proteases and their crosstalks are key factors in its toxicity and accumulation into aggregates. Deciphering the variant composition of tau aggregates is crucial for revealing the aggregation mechanism and understanding the progression of Alzheimer's disease (AD).
[0003] Currently, the identification of tau proteins mainly relies on bottom-up proteomics methods after enrichment. Bottom-up methods analyze intact proteins by digesting them into peptides and then assigning the peptides to proteins, but they cannot analyze at the protein variant level. However, tau proteins have numerous post-translational modifications and truncation sites, resulting in a large number of tau variants. Top-down strategies can study post-translational modifications, sequence variations, truncation, and other information at the intact protein level. Therefore, how to enrich and identify intact tau from actual samples is crucial for the accurate analysis of its variants. Currently, the main strategies for tau enrichment include: (1) obtaining its aggregates using ultracentrifugation; (2) using tau antibodies targeting different sequences for immunoprecipitation. These strategies have the disadvantage of insufficient tau protein specificity or being able to enrich only specific types of tau proteins. Moreover, due to the large molecular weight of tau proteins (>30kDa), they are subject to signal suppression in mass spectrometry response, so there is still a lack of methods for the accurate analysis of tau protein variants.
[0004] This patent addresses the problems of poor specificity in tau protein enrichment and difficulty in identification due to its large molecular weight by developing a method for the enrichment and identification of tau protein variants. Starting from the challenges faced in the enrichment and identification of tau proteins, a method for analyzing tau protein variants was established and applied to tissue samples from neurodegenerative diseases to achieve accurate analysis of tau protein variants. Summary of the Invention
[0005] The purpose of this invention is to provide a method for enriching and identifying tau protein variants in neurodegenerative diseases. The method of this invention solves the problems of poor specificity of tau protein enrichment and difficulty in identification due to its large molecular weight.
[0006] To achieve the above objectives, the technical solution adopted by the present invention specifically includes the following steps:
[0007] (1) Synthesis of recognition peptide functionalized magnetic spheres that specifically enrich tau: For every 20 mg of hydrophilic magnetic spheres with carboxyl groups bonded to their surface (diameter 10-500 nm, carboxyl density >1000 μmol / g, purchased from Aladdin), add 0.01-0.2 mmol of succinimide ester (NHS ester) and 0.01-0.2 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and react at 20-60 °C for 0.5-4 h to activate the carboxyl groups to succinimide ester groups; Remove the reaction solution from the previous step, add 0.001-0.1 mmol equivalent of the recognition peptide TLKIVW, which has good targeting properties to the conserved region of tau protein, and 0.01-0.2 mmol of N,N-diisopropylethylamine (DIEA), and react at 20-60℃ for 0.5-4 h. The amino group in the recognition peptide TLKIVW reacts with the succinimide ester group, thereby binding to the surface of the magnetic beads. After removing the reaction solution, add 1M glycine aqueous solution and react at room temperature for 0.1-2 h to block the unreacted succinimide ester group.
[0008] (2) Targeted enrichment of tau protein: Add sodium lauroyl sarcosinate solution to the protein sample to make the protein concentration 0.1-5 mg / mL and the sodium lauroyl sarcosinate mass concentration 0.1-0.5%; mix the recognition peptide functionalized magnetic beads with the protein at a ratio of 1:10-10:1 and incubate at 0-4℃ for 0.5-4 h; wash the magnetic beads enriched with tau protein with washing solution.
[0009] (3) Top-down identification of tau protein variants: Using 0.1%–10% SDS or 1–6M guanidine hydrochloride as eluent, the tau protein was eluted from the peptide-functionalized magnetic beads at room temperature (300–1400 rpm) for 30 min. The tau protein was then analyzed using liquid chromatography-mass spectrometry (LC-MS), with separation performed using a reversed-phase column and data acquisition based on top-down mass spectrometry using a medium-to-high resolution combined method. Data retrieval of the tau protein was performed using ProsightPD (Thermo Fisher Scientific) to identify tau protein variants.
[0010] (4) Middle-down identification of tau protein variants: Magnetic beads enriched with tau protein were resuspended in enzyme digestion buffer, enzyme was added, and incubation was performed at 20–37°C for 0.1–24 h. Formic acid was then added to bring the final concentration to 1%–5% to terminate the digestion. Separation was performed using a reversed-phase column, and identification was performed using high-resolution mass spectrometry. Data retrieval was conducted using Toppic (https: / / www.toppic.org / software / toppic / index.html) and PEAKS to identify tau protein variant fragments.
[0011] The above method was applied to the analysis of tau protein variants in cells and animal tissues. Addressing the problems of poor specificity in tau protein enrichment and the difficulty in identifying tau protein due to its large molecular weight, this method establishes a robust approach for the enrichment and identification of tau protein variants in neurodegenerative diseases. This method can identify and analyze post-translational modification and truncation information of tau proteins, providing strong technical support and important evidence for studying the biological functions of tau protein variants and their roles in neurodegenerative diseases.
[0012] The present invention has the following advantages:
[0013] (1) The present invention designs and synthesizes a recognition peptide-functionalized magnetic ball for enriching tau protein, which has the characteristics of high enrichment specificity, good selectivity and low non-specific adsorption.
[0014] (2) This invention uses a top-down and middle-down proteomics strategy to identify tau variants. Compared with the traditional bottom-up proteomics method, it can comprehensively study information such as post-translational modifications, sequence variations, and truncation of tau protein at the level of protein variants.
[0015] (3) This invention establishes a process for enriching and identifying tau protein variants, which is universally applicable to cell and tissue samples. Attached Figure Description
[0016] Figure 1 Characterization of the enrichment selectivity of tau protein by peptide-functionalized magnetic beads
[0017] Figure 2 Tau protein variant enrichment and identification process
[0018] Figure 3 Tau protein enrichment in brain tissue of AD mice
[0019] Figure 4 Top-down identification results of tau protein variants
[0020] Figure 5 Middle-down identification results of tau protein variants
[0021] Figure 6 Enrichment of tau protein in tissues of AD mice of different ages
[0022] Figure 7 Post-translational modification profiles of tau protein in tissues of AD mice of different ages Detailed Implementation
[0023] The method provided by the present invention will be described in detail below through examples, but this does not limit the present invention in any way.
[0024] Example 1
[0025] 1. Preparation of peptide-functionalized magnetic beads
[0026] Take 400 μl of carboxyl magnetic beads (50 mg / mL, 100 nm, carboxyl density >1000 μmol / g, purchased from Aladdin), remove the liquid, weigh 6.2 mg EDC (0.04 mmol) and 4.6 mg NHS ester (0.04 mmol), dissolve in 6 mL of N,N-dimethylformamide (DMF), and activate at room temperature with shaking for 1 h, then remove the solution; dissolve 10 mg peptide (0.012 mmol) TLKIVW (synthesized by Qiangyao Biotechnology) in 6 mL of DMF, add 6.6 μl of DIEA, and shake at room temperature for 2 h; wash twice with DMF to remove residual peptides, and then wash once with water. Add 2 mL of water and store at 4 °C.
[0027] Example 2
[0028] Enrichment and selective characterization of peptide-functionalized magnetic beads were performed using a mixture of BSA and tau protein in a buffer solution (containing 20 mM Tris-HCl, 0.1% sodium lauroyl sarcosinate, pH 7.4).
[0029] Solutions of 1 mg / mL bovine serum albumin (BSA, Sigma-Aldrich) and 0.1 mg / mL tau protein (rPeptide) were prepared at protein ratios of 1:0, 10:1, and 100:1 (BSA:tau).
[0030] Mix at a ratio of 1000:1, maintaining a final BSA concentration of 0.5 mg / mL. Add the appropriate amounts of tau protein solution and buffer solution (adjusting the concentration of the mixed solution with buffer solution) to obtain the protein mixture.
[0031] Take 2 mg of the peptide-functionalized magnetic beads prepared in Example 1, wash three times with buffer (containing 20 mM Tris-HCl, 0.1% sodium lauroyl sarcosinate, pH 7.4), add 800 μl of the prepared protein mixture, and incubate at 4°C for 2 h by rotation. Separate the magnetic beads from the supernatant by magnetic attraction, and wash with washing buffer (containing 20 mM Tris-HCl, 300 mM NaCl, 0.1% sodium lauroyl sarcosinate, pH 7.4).
[0032] Wash the magnetic beads three times and discard the supernatant. Finally, add 100 μl of 1% SDS and shake at 1400 rpm for 30 min at room temperature to elute the tau protein from the magnetic beads.
[0033] Protein mixtures at four different BSA:tau ratios (L), enriched supernatant (F), and elution buffer (E) were loaded onto an SDS-PAGE gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue. Results are as follows: Figure 1 The results at a BSA:tau ratio of 1:0 showed no bands in the eluent, indicating low non-specific adsorption of proteins by the functionalized magnetic beads. However, at BSA:tau ratios of 10:1, 100:1, and 1000:1, tau bands were observed in the 100:1 eluent, demonstrating that the functionalized magnetic beads could enrich tau protein in 100-fold amounts of other proteins.
[0034] Example 3
[0035] according to Figure 2 The enrichment and identification process of tau protein in tissue samples involves using the recognition peptide-functionalized magnetic beads prepared in Example 1 for the enrichment and identification of tau protein. The steps are as follows:
[0036] 1. Enrichment of tau protein in brain tissue of P301S mice
[0037] Add 50 mg of mouse brain tissue to 500 μl of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM EDTA), homogenize the tissue at -40 °C using a low-temperature tissue homogenizer, centrifuge at 5000 g for 10 min, and collect the supernatant. Add 1% sodium lauroyl sarcosinate solution (containing 20 mM Tris-HCl, 1% sodium lauroyl sarcosinate, pH 7.4) to a final concentration of 0.1%, and incubate at 4 °C for 30 min to obtain the protein solution.
[0038] Tau protein was enriched in P301S mouse tissue using peptide-functionalized magnetic beads: 2 mg of peptide-functionalized magnetic beads prepared in Example 1 were taken and washed three times with lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 2 mM EDTA). 800 μl of protein solution was added, and the mixture was incubated at 4 °C for 2 h by rotation. The magnetic beads were separated from the supernatant by magnetic attraction. The magnetic beads were washed three times with washing buffer (containing 20 mM Tris-HCl, 300 mM NaCl, 0.1% sodium lauroyl sarcosinate, pH 7.4). The supernatant was discarded, and magnetic beads enriched with tau protein were obtained.
[0039] Elution: Finally, add 100 μl of 1% SDS to the magnetic beads enriched with tau protein and shake at 1400 rpm for 30 min at room temperature to elute the tau protein from the magnetic beads. Figure 3Western blot analysis showed that using tau-5 antibody as the primary antibody, tau bands were visible in the elution sample, indicating that the recognition peptide-functionalized magnetic beads successfully enriched tau protein in the tissue sample.
[0040] 2. Top-down identification of tau protein in brain tissue of P301S mice
[0041] Tau protein in P301S mouse tissue was enriched using recognition peptide-functionalized magnetic beads. In the elution step, 100 μl of 6M guanidine hydrochloride was added to the magnetic beads enriched with tau protein and shaken at 1400 rpm for 30 min at room temperature to elute the tau protein from the magnetic beads, thus obtaining the sample solution.
[0042] The sample solution was loaded (offline) onto an 8 cm long pre-column (C1 functionalized ethyl-bridged hybrid monolithic column, preparation process referred to the literature Wang, C., et al., Ethane-Bridged Hybrid Monolithic Column with Large Mesopores for Boosting Top-Down Proteomic Analysis. Anal Chem, 2022, 94(16): p.6172-6179.).
[0043] The pre-column was then washed with 0.1% FA to remove 6M guanidine hydrochloride. The pre-column was then connected to a C1-functionalized ethyl-bridged hybrid monolithic column (the pre-column outlet was connected to the sample inlet of the C1-functionalized ethyl-bridged hybrid monolithic column) for intact protein separation. A combined medium- and high-resolution mass spectrometry method was used for identification: a medium-resolution mass spectrometer (7500 nm) was used for the first-stage mass spectrometry, and a high-resolution mass spectrometer (60000 nm) was used for the second-stage mass spectrometry. Data retrieval of tau protein was performed using ProsightPD (Thermo Fisher Scientific) to identify tau protein variants.
[0044] like Figure 4 Four tau protein variants were identified, each carrying a phosphorylation site associated with AD, and co-occurrence of PTM at different sites was observed. Among them, an N-terminal truncated variant (1-221) and a C-terminal truncated variant (193-412) that have not been reported in the literature were identified.
[0045] 3. Middle-down identification of tau protein in P301S mouse brain tissue
[0046] Tau protein in P301S mouse tissues was enriched using peptide-functionalized magnetic beads. 90 μl of incubation buffer (50 mM Tris-HCl, 5 mM CaCl2, 2 mM EDTA, pH 7.6) was added to the tau-enriched beads. ArgC protease was added at a 1:20 enzyme-to-protein ratio, followed by 10 μl of activation buffer (50 mM Tris-HCl, 50 mM DTT, 2 mM EDTA, pH 7.6). The mixture was incubated at 37°C for 15 min, and then acidified with 100 μl of 10% formic acid to terminate the enzymatic digestion. The supernatant was collected after centrifugation as the tau protein digestion solution. The tau protein was separated and identified using a C18-functionalized ethyl-bridged hybrid monolithic column (the matrix preparation process was described in the literature Wang, C., et al., Ethane-Bridged Hybrid Monolithic Column with Large Mesopores for Boosting Top-Down Proteomic Analysis. Anal Chem, 2022, 94(16): p.6172-6179. To achieve C18 functionalization, the functionalization group modification step was modified: 20% (v / v) octadecyl dimethylamine dimethylsilane dissolved in anhydrous toluene was continuously pumped into the monolithic column matrix under nitrogen pressure at 60°C for 24 h, followed by blocking with 20% (v / v) N-(trimethylsilyl)imidazolium at 60°C for 24 h to complete the functionalization modification). High-resolution mass spectrometry was used for separation and identification, with both primary and secondary mass spectrometry at 60,000 resolution. Tau protein fragments were identified by data search using PEAKS.
[0047] like Figure 5 The identified tau protein fragment sequence coverage was 65%, including post-translational modifications such as deamidation, methylation, oxidation, phosphorylation, and ubiquitination. Multiple post-translational modifications were observed in the same fragment, and different variants of tau were detected.
[0048] Example 4
[0049] according to Figure 2 The enrichment and identification process of tau protein involved using the recognition peptide-functionalized magnetic beads prepared in Example 1 to enrich and identify tau protein in hippocampal tissue samples from 3-, 6-, and 9-month-old AD mouse models (the process and conditions were the same as in Example 3). Figure 6 Western blot analysis showed that using tau-5 antibody as the primary antibody, tau bands were observed in the elution sample, indicating that the peptide-functionalized magnetic beads successfully enriched tau protein in hippocampal tissue samples from 3-, 6-, and 9-month-old AD mice. Figure 7Using a middle-down identification method, multiple phosphorylation sites reported in the literature were covered; with increasing age, the C-terminal PTM type and site of tau protein changed significantly; multiple combined variants of phosphorylation, methylation, ubiquitination and deamidation were observed.
Claims
1. A method for enrichment and identification of tau protein variants in neurodegenerative diseases, characterized by: Includes the following steps: (1) Prepare magnetic beads with recognition peptides that have good targeting properties to conserved tau regions; (2) Mix the sample with the recognition peptide-functionalized magnetic beads to target and enrich tau protein; (3) Obtain intact tau protein by elution and perform top-down separation and identification of tau protein variants; (4) Obtain fragmented tau protein by restriction enzyme digestion and perform middle-down identification of tau protein variants.
2. The method as described in claim 1, characterized in that: The synthesis process of the recognition peptide-functionalized magnetic spheres described in step 1) includes: adding 0.01-0.2 mmol of succinimide ester (NHS ester) and 0.01-0.2 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to every 20 mg of hydrophilic magnetic spheres with surface-bonded carboxyl groups, reacting at 20-60 °C for 0.5-4 h to activate the carboxyl groups into succinimide ester groups; removing the surface-bonded carboxyl groups. In a one-step reaction solution, add 0.001-0.1 mmol equivalent of the recognition peptide TLKIVW, which has good targeting properties to the conserved region of tau protein, and 0.01-0.2 mmol of N,N-diisopropylethylamine (DIEA). React at 20-60℃ for 0.5-4 h. The amino group in the recognition peptide TLKIVW reacts with the succinimide ester group, thereby binding to the surface of the magnetic beads. After removing the reaction solution, add 0.5-1.5M glycine aqueous solution and react at room temperature for 0.1-2 h to block the unreacted succinimide ester group.
3. The method as described in claim 1, characterized in that: The process of targeted enrichment of tau protein described in step 2) includes: adding sodium lauroyl sarcosinate solution to the protein sample to make the protein concentration 0.1-5 mg / mL and the sodium lauroyl sarcosinate mass concentration 0.1-0.5%; mixing the recognition peptide-functionalized magnetic beads with the protein at a ratio of 1:10-10:1 and incubating at 0-4℃ for 0.5-4 h; and washing the magnetic beads enriched with tau protein with a washing solution.
4. The method as described in claim 3, characterized in that: The sodium lauroyl sarcosinate solution is a buffer solution containing 0.1-10% sodium lauroyl sarcosinate, using Tris-HCl buffer as the solvent; The washing solution is one or more of the following: sodium lauroyl sarcosinate solution, 150-900mM sodium chloride solution, or Tris-HCl buffer.
5. The method as described in claim 1 or 3, characterized in that: The elution method described in step 3) is to use 0.1-10% sodium dodecyl sulfonate (SDS) or 1-6M guanidine hydrochloride as the eluent, and shake at room temperature for 20-40 minutes at 300-1400 rpm.
6. The method as described in claim 1, characterized in that: The top-down identification method for tau protein variants described in step 3) includes: separating the intact tau protein using a reversed-phase chromatography column and identifying it using a top-down mass spectrometry method combining medium and high resolution. The reversed-phase chromatographic column comprises one or more functionalized monolithic reversed-phase columns of C1-C8 with a length of 20-100 cm and an inner diameter of 50-100 μm. The aforementioned top-down mass spectrometry identification method combining medium and high resolution uses medium resolution (7500-15000) for primary mass spectrometry detection and high resolution (60000-240000) for secondary mass spectrometry detection.
7. The method as described in claim 1, characterized in that: The tau protein fragmentation method described in step 4) is as follows: resuspend the magnetic beads enriched with tau protein using an enzyme digestion buffer, add enzyme, incubate at 20-37℃ for 0.1-24h, and then add formic acid to make the final concentration 1-5% to terminate the enzyme digestion. The enzyme type includes one or more of ArgC, OmpT, and LysC.
8. The method as described in claim 1, characterized in that: The middle-down identification method for tau protein variants described in step 4) includes: separating fragmented tau using a reversed-phase chromatography column; and identifying it using high-resolution middle-down mass spectrometry. The reversed-phase chromatographic column comprises one or more functionalized monolithic reversed-phase columns of C1-C18 with a length of 20-100 cm and an inner diameter ranging from 50-100 μm. The aforementioned high-resolution mass spectrometry identification techniques, MS and MS 2 All of them used a high-resolution acquisition method of 30,000-240,000.
9. The method as described in claim 3, characterized in that: Cells and / or biological tissues are broken up and homogenized to obtain protein samples.