A method and kit for in vitro detection of alpha-synuclein o-glcnaclylation modification

Abstract

The application discloses an in-vitro detection method and kit for O-GlcNAc glycosylation modification of alpha-synuclein, and relates to the field of biochemical detection. Y289L The method comprises the following steps: extracting protein in a sample to be detected, adding UDP-GalNAz substrate beta4GalT Y289L The mutant enzyme and the reaction are carried out to obtain a protein system; DBCO-mPEG is added to the protein system, and the reaction product is subjected to electrophoretic separation; and according to an electrophoretogram, the proportion of the protein subjected to glycosylation modification and the molecular weight distribution thereof are calculated. The application marks specific molecular weight "tags" for O-GlcNAc glycosylation proteins, and the glycosylation-modified proteins are recognized by alpha-synuclein specific antibodies to show specific molecular weight sizes; and the alpha-synuclein polymerization states, such as dimers and polymers, subjected to O-GlcNAc glycosylation modification can be determined and quantified according to protein standards.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, and in particular to an in vitro detection method and kit for α-synuclein O-GlcNAc glycosylation modification. Background Technology

[0002] α-synuclein (α-syn) aggregation is a key molecular process in the progression of various neurodegenerative diseases, including Parkinson's disease, multiple system atrophy (MSA), and Lewy body dementia (LDB). α-syn aggregation is a progression from low-molecular-weight monomers to dimers, oligomers, and multimers. A dynamic equilibrium exists between the early aberrantly folded monomers, dimers, and oligomers, and the allosteric changes between these structural forms are reversible. Under pathological or microenvironmental abnormalities, oligomeric components rapidly aggregate to form high-molecular-weight, insoluble toxic fibers, causing cellular damage. Studies have shown that α-synuclein undergoes extensive post-translational modifications (PTMs), such as ubiquitination, phosphorylation, and glycosylation, which significantly influence its pathological aggregation and cytotoxicity.

[0003] O-GlcNAc glycosylation is an essential post-translational modification in mammals. It involves the addition of functionalized O-linked β-N-acetylglucosamine monomers (GlcNAc) to the serine and threonine side chains of proteins under the catalysis of O-GlcNAc transferase (OGT) or the removal of functionalized O-linked β-N-acetylglucosamine monomers under the catalysis of O-GlcNAc glycosidase (OGA). Figure 1O-GlcNAc glycosylation is dynamic and inducible, playing a crucial role in cellular signaling pathways, cell fate determination, transcription, immune responses, and responses to cellular stressors. Studies have shown that O-GlcNAc glycosylation of α-synuclein significantly inhibits α-synuclein aggregate formation and reduces phosphorylation of the aggregate marker α-synuclein pS129. For example, by artificially modifying the amino acid sequence of α-synuclein with O-GlcNAc glycosylation using in vitro chemical synthesis, site-specific O-GlcNAc glycosylation at threonine residue 72 (gT72) or serine residue 87 (gS87) has been found to inhibit α-synuclein aggregation. Introducing PFFs (progeny fine-grained fibers) from site-specifically O-GlcNAc-glycosylated α-synuclei into primary neurons revealed significant regression in aggregation. Furthermore, O-GlcNAc glycosylation of α-synuclei reduced neuronal Capcase-3 enzyme activation and inhibited premature neuronal death. Therefore, O-GlcNAc glycosylation modification can inhibit α-synuclein aggregation and exert a protective effect on damaged neurons. Developing a detection method for α-synuclein glycosylation modification has important practical and translational significance.

[0004] Although O-GlcNAc glycosylation is conserved across species, methods for detecting and quantifying specific protein modifications remain lacking, posing a challenge to the study of the function of O-GlcNAc glycosylation modifications in proteins. The main reasons are (i) a lack of effective tools and techniques to study O-GlcNAc glycosylation in specific proteins; and (ii) the fragment-specific nature of O-GlcNAc glycosylated proteins. Adding sugars to proteins or peptide chains does not alter the molecular weight or isoelectric point of O-GlcNAc glycosylated proteins, rendering conventional techniques such as gel electrophoresis, two-dimensional gel electrophoresis, and high-performance liquid chromatography (HPLC) impractical. For these reasons, effective methods for detecting the dynamic changes in O-GlcNAc glycosylation modifications of α-synuclein proteins in tissue cells, especially modifications in the α-synucleus aggregated state, are still lacking.

[0005] Currently widely used methods for detecting O-GlcNAc protein modification include: radionuclide labeling, specific antibody methods, lectin capture methods, and chemical enzyme metabolic labeling methods. These methods have specific applicable conditions and some key limitations in detecting modifications in the α-synaptic nucleus aggregation state.

[0006] Early radionuclide labeling methods required handling expensive radioactive materials and typically involved exposure for days to months. Furthermore, similar to labeling methods for radionuclides such as tritium, antibody- and lectin-based techniques are often insufficiently sensitive in low-abundance or limited-sample conditions, failing to detect low-abundance O-GlcNAc modifications in proteins.

[0007] Pan-O-GlcNAc antibody detection lacks specificity, easily leading to errors and failing to detect single specific proteins. For example, Western blotting using a CTD110.6 spectrometer observed an increase in O-GlcNAc levels after nutrient deprivation, but actual detection revealed this was due to an increase in truncated N-glycans. Because GlcNAc residues are neutral sugars with poor immunogenicity, and due to a lack of in-depth understanding of O-GlcNAc glycosylation sequences, the development of selective antibodies against α-synuclein proteins, especially site-specific antibodies, is limited. Antibodies recognize unique GlcNAc epitopes and may cross-react with other sugar fragments; furthermore, due to varying antibody selectivity, their specificity for single proteins is poor, limiting the application of detecting O-GlcNAc proteins and obtaining validation results via mass spectrometry (MS). Additionally, another drawback of antibody methods is the high difficulty in antibody purification, which also limits their development as a method for detecting O-GlcNAc modification in α-synuclein proteins.

[0008] WGA (Leechin Capture Gel) is another commonly used method for detecting O-GlcNAc glycosylation modification of specific proteins, and sWGA with higher affinity has been developed. However, this method still does not completely solve the problem of low detection sensitivity. Furthermore, the mixture of lectin WGA and acrylamide gel is susceptible to environmental pollutants such as heavy metals, and copolymerization is difficult. Lectin-conjugated polyacrylamide gel electrophoresis can identify glycosylated and non-glycosylated proteins using specific antibodies against α-synuclein, but because this method lacks clear protein standards, it cannot detect O-GlcNAc glycosylation modification in protein polymeric states (such as dimers, multimers, etc.). Secondly, because WGA binds not only to N-acetylglucosamine residues but also non-specifically to sialic acid, it is prone to detection errors.

[0009] Given the limitations of the above methods, it is crucial to develop a highly sensitive and specific method capable of assessing α-synuclein O-GlcNAc glycosylation modifications under different states. Summary of the Invention

[0010] Objectives of the invention: The first objective of this invention is to provide a highly sensitive, specific method for detecting α-synuclein O-GlcNAc glycosylation modification that can distinguish protein polymerization states; the second objective of this invention is to provide a kit for detecting α-synuclein O-GlcNAc glycosylation modification.

[0011] To achieve the aforementioned primary objective, the technical solution of the α-synuclein O-GlcNAc glycosylation modification detection method provided by this invention is as follows:

[0012] The detection method described in this invention is an in vitro detection method, which includes the following steps:

[0013] (1) Extract the protein from the sample to be tested, and add UDP-GalNAz (uridine diphosphate-N-azidoacetylgalactosamine) substrate and β4GalT to the protein. Y289L The mutant enzyme performs the reaction; if the protein in the sample to be tested has O-GlcNAc glycosylation modification, the mutant enzyme transfers the GalNAz (N-azidoacetylgalactosamine) group to the O-GlcNAc residue to obtain the target protein labeled with the azide group.

[0014] (2) DBCO-mPEG is added to the protein system obtained in step (1) to carry out a copper-free click chemical reaction; if the protein in the sample to be tested has O-GlcNAc glycosylation modification, DBCO-mPEG will undergo a cycloaddition reaction with the azide group to obtain the PEGylated target protein.

[0015] (3) The reaction products obtained in step (2) are separated by SDS-PAGE electrophoresis; if the protein in the sample to be tested has O-GlcNAc glycosylation modification, then in the electrophoresis pattern, the protein modified by O-GlcNAc glycosylation shows a lag in migration rate compared with the protein without O-GlcNAc glycosylation modification.

[0016] (4) Based on the gray values ​​of the protein bands without O-GlcNAc glycosylation and the protein bands with GlcNAc glycosylation in the electrophoresis pattern, calculate the proportion of proteins with O-GlcNAc glycosylation and their molecular weight distribution in the sample to be tested.

[0017] In step (1), the sample to be tested is selected from: cell lysis buffer, tissue homogenate, cerebrospinal fluid, or serum. β4GalT Y289L Mutant enzymes are engineered enzymes based on the soluble catalytic domain of β1,4-galactosyltransferase I (β4Gal-T1) (typically with the N-terminal transmembrane region removed, starting from approximately amino acid 130), and with a site-directed mutation of tyrosine (Tyr) at position 289 in the catalytic core to leucine (Leu).

[0018] In step (2), the molecular weight of DBCO-mPEG added to the protein system is adjusted according to the polymerization state of α-synuclein in the sample to be tested. If the α-synuclein in the sample to be tested is a monomer, DBCO-mPEG with a molecular weight greater than or equal to 10 kDa is added to the protein system; if the α-synuclein in the sample to be tested is a polymer, DBCO-mPEG with a molecular weight of 4-6 kDa is added to the protein system.

[0019] In step (3), the reaction product is separated by SDS-PAGE electrophoresis to obtain the target protein, and the target protein is detected by immunoblotting to identify whether the protein is an α-synuclein.

[0020] In step (4), the molecular weight distribution or aggregation form of α-synuclein modified with O-GlcNAc is identified in the electrophoresis pattern based on the reference of protein molecular weight standards. The protein bands corresponding to α-synuclein modified with O-GlcNAc are cut and the target α-synuclein is enriched. The O-GlcNac modification sites of the target α-synuclein are identified by high-performance liquid chromatography-mass spectrometry.

[0021] The detection method of this invention is used to screen drug candidate molecules that regulate the modification level of α-synuclein O-GlcNAc.

[0022] To achieve the second objective mentioned above, the technical solution of the reagent kit provided by the present invention is as follows:

[0023] The kit for detecting α-synuclein O-GlcNAc glycosylation modification according to the present invention comprises: β4GalT Y289L Mutant enzyme, UDP-GalNAz substrate, and DBCO-mPEG reagent.

[0024] The kit also includes protein extraction reagents, protein precipitation reagents, and labeling buffers, wherein the labeling buffers contain 10 mM TEA, 150 mM NaCl, and 1% SDS.

[0025] Invention Principle: O-GlcNAc modification (N-acetylglucosamine modification) typically occurs on serine / threonine residues, and the modified group has a small molecular weight, making it difficult to distinguish modified from unmodified proteins by migration differences in conventional SDS-PAGE electrophoresis. This invention employs a strategy of "chemical enzymatic labeling combined with mass tag weight gain": First, β4GalT... Y289LThe mutant enzyme and UDP-GalNAz substrate specifically introduce GalNAz with an azide group (-N3) onto the O-GlcNAc residues, transforming the previously "hidden" glycosylation modification into a "dominant" target with bioorthogonal reactivity. Subsequently, DBCO-mPEG is added, utilizing its terminal DBCO group to undergo an efficient copper-free click chemistry reaction with the azide group on the protein, thereby covalently linking a PEG tag with a significant molecular weight increase to the O-GlcNAc-modified protein. Then, the protein is analyzed by SDS-PAGE. Gel electrophoresis separation was performed, and due to the introduction of the PEG tag, the modified protein showed a significant lag in electrophoretic migration compared to the unmodified protein. Finally, immunoblotting was performed using α-synuclein-specific antibodies. Based on protein molecular weight standards, it was possible not only to identify and distinguish different polymerization forms of α-synuclein modified with O-GlcNAc (such as monomers, dimers, and multimers), but also to achieve precise quantitative analysis of the glycosylation level and polymerization state of α-synuclein O-GlcNAc by comparing the gray values ​​of the bands before and after modification.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The method first unbiasedly labels O-GlcNAc glycosylated proteins with specific molecular weight "tags". The labeled proteins migrate slowly under SDS-PAGE gel electrophoresis, and the glycosylated modified proteins recognized by α-synuclein specific antibodies will show specific molecular weights. The polymerization state of α-synuclein modified by O-GlcNAc glycosylation, such as dimers and multimers, can be determined and quantified according to protein standards. Therefore, the method can simultaneously detect the O-GlcNAc glycosylated and non-O-GlcNAc glycosylated modification forms of α-synuclein and perform quantitative analysis. (2) The method allows for adjustment of the α-synuclein detection antibody according to detection needs and sample sources; and the molecular weight of the added tag can be adjusted according to the state of the target α-synuclein to adapt to the detection requirements. For example, for detecting α-synuclein monomers modified by O-GlcNAc glycosylation, a tag of >10 kDa can be added, and for polymers, a small molecular weight tag such as 5 kDa can be added to achieve effective separation by SDS-PAGE. (3) The method is compatible. Based on the preliminary detection results, the protein containing O-GlcNAc compounds of interest can be dissolved from the gel, and high-performance liquid chromatography-protein spectroscopy-MS / MS technology can be used to further verify and identify the exact modification sites on the α-synuclein peptides modified by O-GlcNAc, which helps in the downstream analysis of the glycosylation kinetics and function of the target protein. This invention identified 4 glycosylation sites by MS / MS mass spectrometry, of which 2 were newly identified sites. (4) The enzymes and reaction reagents used in this method are all conventional reagents, β4GalT Y289LThere are also commercial reagent alternatives (such as Yinjia Bio's YJ-O-311), and this method is simple and easy to master, and can detect α-synuclein proteins that have undergone O-GlcNAc glycosylation with high specificity and sensitivity. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the protein O-GlcNAc cycle catalyzed by OGA and OGT enzymes, where UDP-GlcNAc is uridine diphosphate-N-acetylglucosamine, OSMI-1 is an O-GlcNAc transferase inhibitor, and O-GlcNAcase (OGA) is an O-linked β-N-acetylglucosamine monomer.

[0028] Figure 2 This is a flowchart of the detection method for α-synuclein O-GlcNAc glycosylation modification of the present invention;

[0029] Figure 3 A schematic diagram illustrating the process of chemical enzyme coupling with PEG quality tag labeling and the detection of O-GlcNAc modified α-synuclein by Western blotting.

[0030] Figure 4 Construction of the pET-23a-β4GalTY289L recombinant protein expression vector and the amino acid sequence and mutation site of the inserted gene fragment;

[0031] Figure 5 IPTG-induced expression of pET-23a-β4GalTY289L plasmid, SDS-PAGE separation of recombinant protein, and identification by Coomassie Brilliant Blue R-250 staining;

[0032] Figure 6 Western blot results of pEGFP-SNCA transfected cells treated with OSMI-1 and Thiamet-G (TMG), and detection of protein O-GlcNAc modification levels with RL-2 antibody;

[0033] Figure 7 The results of Western blot analysis of pEGFP-SNCA transfected SH-SY5Y cells with O-GlcNAc were shown. In the figure, A is a representative image of the Western blot detection results of O-GlcNAc-modified α-synuclein; B is a quantitative analysis of the percentage (%) of O-GlcNAc-modified α-synuclein in the total protein (actually the protein at ~43 kDa) detected by Western blot.

[0034] Figure 8 IPTG-induced expression of pET-21a-SNCA plasmid, SDS-PAGE separation of recombinant α-synuclein, and identification by Coomassie Brilliant Blue R-250 staining;

[0035] Figure 9 The Western blot results are for PFFs generated by shaking recombinant α-synuclein and then subjected to sonication.

[0036] Figure 10 Western blot results of PFF seeding and Thiamet-G (TMG) treatment of SH-SY5Y cells;

[0037] Figure 11 The results of Western blot analysis of SH-SY5Y cells treated with PFFs after O-GlcNAc regulation are shown. In the figure, A is a representative image of the Western blot detection results of O-GlcNAc-modified α-synuclein; B is a quantitative analysis of the percentage (%) of O-GlcNAc-modified α-synuclein in the total protein (actually the protein at ~35 kDa) detected by Western blot.

[0038] Figure 12 Results of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) of O-GlcNAc glycosylation (G) modified peptides (A) and analysis of amino acid sequence conservation among species (B).

[0039] Figure 13 MS / MS mass spectra of O-GlcNAc glycosylation modification at S42 of α-synuclein;

[0040] Figure 14 MS / MS mass spectra of O-GlcNAc glycosylation modifications at T44, T54 and T59 of α-synuclein;

[0041] Figure 15 MS / MS mass spectrum of O-GlcNAc glycosylation modification at T54 of α-synuclein. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0043] like Figure 2-3 As shown, this invention introduces a chemical handle via enzymatic methods, and then creates significant molecular weight differences by clicking on chemically linked high-molecular-weight tags, thereby achieving visual differentiation. This invention uses the Y289L mutant of bovine β-1,4-galactosyltransferase 1 (β4GalT). Y289LThis invention utilizes N-azidogalactosamine (GalNAz) for unbiased labeling of O-GlcNAc glycosylated proteins. This method allows for specific, unbiased, and global labeling of specific modification sites on O-GlcNAc glycosylated proteins. Then, a azido-acetylene cycloaddition "click" reaction is used to attach a polyethylene glycol (mPEG) tag of a certain molecular weight to the O-GlcNAc glycosylated protein, adding a high-molecular-weight azide "chemical handle" to the glycosylated protein. To achieve efficient separation of α-synuclein proteins in different conformational states of O-GlcNAc glycosylation, this invention employs DEPC-mPEG with a molecular weight of 4-6 kDa (since the molecular weight of α-synuclein monomers is approximately 14-15 kDa, 10 kDa DEPC-mPEG can also be used for unlabeled monomers in conventional Western blotting). This invention uses SDS-PGAE to separate labeled and unlabeled proteins, which are then transferred to a PVDF membrane. α-synuclein proteins are then recognized using specific antibodies in conjunction with Western blotting and immunoblotting. ECL (Extracorporeal Chromatography-Liquid Crystallography) visualizes labeled proteins. By comparing the detected band positions with the molecular weights of standard proteins, α-synuclein proteins undergoing O-GlcNAc glycosylation and non-glycosylation modifications can be identified and subjected to quantification analysis. Simultaneously, the O-GlcNAc glycosylation state of α-synuclein proteins can be determined based on the molecular weight migration position and comparison with standard molecular weights. This method can also be combined with high-performance liquid chromatography-mass spectrometry (MS / MS) to pinpoint the exact serine and threonine residues on O-GlcNAc-modified α-synuclein peptides.

[0044] Example 1

[0045] β4GalT Y289L Enzyme preparation includes the following steps:

[0046] 1) The gene sequence corresponding to amino acids 130-402 of the catalytic domain of the β4GalT enzyme (Protein ID: NP_803478.1) was cloned, and the 289th position was mutated from Tyr to Leu to enhance expression and catalytic efficiency. After digestion with BamHI at the 5' end and XhoI at the 3' end, the sequence was inserted into the backbone of the prokaryotic expression vector pET-23a to construct pET-23a-β4GalT. Y289 Recombinant protein expression vector ( Figure 4 ).

[0047] 2) Use 1 μL of β4GalT Y289L The expression plasmid (10 ng / μL) was transformed into 50 μL of BL21 (DE3) competent Escherichia coli, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37 °C.

[0048] 3) Pick a single colony and inoculate it into 5 mL of LB liquid medium containing 100 μg / mL ampicillin. Incubate overnight at 37°C with shaking at 220 rpm.

[0049] 4) Distribute 60 ml of the culture evenly into 1 L of LB liquid medium containing 100 μg / mL ampicillin. Incubate at 37 ℃ and 220 rpm with shaking for 2-3 h until the OD (A600) reaches 0.7.

[0050] 5) Add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM to induce protein expression. Continue incubation at 37 ℃ and 220 rpm with shaking for 4 h or overnight.

[0051] 6) Centrifuge 8000 g of the shaken bacterial culture at 4 °C for 10 min, discarding the supernatant completely. Determine β4GalT Y289L Protein expression at different induction time points (e.g., no induction, IPTG induction at 4, 8, and 16 h). Detection was performed using Coomassie Brilliant Blue staining, with molecular weight included as a reference.

[0052] 7) Resuspend the bacteria from 1 L of culture medium into 30 ml of resuspension buffer (25% (w / v) sucrose, 0.1% Triton X-100, 1 mM EDTA, 1* phosphate buffer PBS pH 7.4), and sonicate on ice for lysis. Perform intermittent sonication in 10 ml portions: sonicate on for 30 seconds, pause for 30 seconds, and repeat the process for a total sonication time of 12 minutes.

[0053] 8) Dilute the bacterial lysate to 200 ml with PBS containing 1 mM EDTA, and centrifuge at 15,000 g and 4 °C for 30 min.

[0054] 9) Discard the supernatant, add the same volume of resuspension buffer, and resuspend and wash the precipitate 5 times. Ensure complete resuspension of the precipitate in each washing step to achieve β4GalT. Y289L Effective purification. After repeated washing, the precipitate should change from yellow to milky white. If necessary, the precipitate can be refrigerated overnight at 4 °C. After the final centrifugation, resuspend the precipitate in 50 ml of PBS containing 1 mM EDTA and centrifuge at 14,000 g for 30 min.

[0055] 10) Discard the supernatant and repeat step 9) to remove the residual detergent (Triton X-100) again.

[0056] 11) Discard the supernatant and resuspend the precipitate in 24 mL of lysis buffer containing 8 M urea and 0.3 M Na2SO3.

[0057] 12) Add 2 mL of the S-sulfonating agent 4-nitrobenzyl-2-oxa-1,3-diazole-4-thiol (NTSB) and stir vigorously until the solution turns pale yellow. After adding NTSB, the solution should turn reddish-orange. Continue stirring until the solution turns pale yellow again; the reaction usually takes about 45 minutes.

[0058] 13) Add 250 mL of pre-cooled ultrapure water (MilliQ-H2O) to precipitate the protein. Centrifuge at 15,000 g, 4 °C for 30 min. Discard the supernatant and resuspend the precipitate in 250 mL of pre-cooled MilliQ-H2O. Resuspend 2-3 times to remove residual NTSB sulfonating agent.

[0059] 14) Discard the supernatant, dilute the precipitate with 8 M urea and MilliQ-H2O, and dissolve overnight at 4 °C. Quantify to a final concentration of 1 mg / ml.

[0060] 15) Dilute the protein diluent 10-fold with pre-cooled renaturation buffer to a concentration of approximately 0.1 mg / ml. During the mixing process, slowly add the renaturation buffer dropwise to the protein diluent 10 times, and gently stir overnight at 4 ℃ or renaturate for 48 h without stirring.

[0061] 16) Dialyze with 2 L of dialysis buffer for 36 h. Because a large amount of protein will precipitate during dialysis, centrifuge at 10,000 g for 10 min after dialysis to remove the protein precipitate.

[0062] 17) Concentrate to 5 ml using a centrifugal filtration device, to a final concentration of 2 mg / mL, and store at 4 ℃ for later use.

[0063] Take a portion of the concentrated solution obtained in step 17), dissolve it in the loading buffer, separate it with an SDS-PAGE gel, and then stain it with Coomassie Brilliant Blue R-250 to identify β4GalT. Y289L Recombinant proteins. For example... Figure 5 As shown, by comparing the results before and after IPTG induction, it was found that in the lanes before IPTG induction, there were only weak background bands at the target location. In the lanes at 4 h, 8 h, and 16 h, the corresponding β4GalT bands were significantly reduced. Y289L A band that gradually thickens and darkens is observed at the molecular weight position; β4GalT Y289L The highest expression level was observed 16 h after IPTG induction; therefore, the protein obtained at this induction time was used as the β4GalT protein for subsequent experiments. Y289L Enzymes.

[0064] Example 2

[0065] The steps for O-GlcNAc glycosylated protein-catalyzed coupling of GalNAz and DBCO-mPEG are as follows:

[0066] 1. Protein sample preparation

[0067] Cells or tissues were placed in a buffer solution (25 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 20 μM hexosinosinase inhibitor PUGNAc, 1 mM PMSF) and homogenized on ice. After incubation on ice for 30 min, the cells were centrifuged (12000 rpm, 4 °C for 10 min), and the supernatant (approximately 200 μL) was collected for later use.

[0068] 2. Protein precipitation

[0069] Add 600 μL of methanol, 150 μL of chloroform, and 400 μL of ultrapure water sequentially to 200 μL of the supernatant obtained in step (1). Mix well and centrifuge the resulting mixture (12000 rpm, 15 ℃ for 15 min). Discard the supernatant; the protein should be located at the interface. Add 450 μL of methanol to wash and mix well. Centrifuge again (12000 rpm, 15 ℃ for 15 min) and discard the supernatant. Air dry the obtained sample at room temperature for 5 min.

[0070] After air-drying, the precipitate was dissolved and mixed with 40 μL of resuspension buffer A (20 mM 4-hydroxyethylpiperazine ethanesulfonic acid HEPES (pH 7.9), 1% SDS), and then placed on ice for 3 min. The protein concentration was determined by BCA method, and each sample was diluted to 2.5 μg / μL with resuspension buffer A.

[0071] 3. Gal-T1 Y289L enzyme catalyzes GalNAz coupling

[0072] In step 2, 49 μL of ultrapure water was added to the final sample and mixed well. Then, 83 μL of labeling reaction buffer (2.5×, 125 mM NaCl, 50 mM HEPES, 5% NP-40, pH 7.9), 11 μL of MnCl2 (100 mM), and 10 μL of UDP-GalNAz working solution were added sequentially and mixed well to obtain a suspension. 50 μL of the above suspension was taken as an unlabeled control. 7.5 μL of Gal-T1 (Y289L) was added to the remaining suspension and mixed well. At this point, the sample volume in the tube was ~150 μL. The suspension was labeled and reacted in a light-protected environment at 4 ℃ for 20 h. The GalNAz-coupled sample was obtained.

[0073] 4. DBCO-mPEG quality-tagged enzyme chemical labeling

[0074] In step 3, 7.5 μL of iodoacetamide (600 mM) was added to the GalNAz-conjugated sample, and the mixture was incubated for 30 min in a light-protected suspension apparatus. The protein was then precipitated using a methanol / chloroform extraction method. 600 μL of methanol was added to the purified sample, and the mixture was stirred. 150 μL of chloroform and 400 μL of ultrapure water were added to the mixed solution, and the mixture was stirred. The mixture was centrifuged (12000 rpm, 15°C for 15 min), and the supernatant was discarded; the protein was located at the interface. Then, 450 μL of methanol was added for washing, and the mixture was stirred. The mixture was centrifuged (12000 rpm, 15°C for 15 min), and the supernatant was discarded. The remaining protein was air-dried at room temperature for 5 min. After air-drying, add 100 μL of mass spectrometry labeling buffer (10 mM triethanolamine, pH 7.4; 150 mM NaCl; 1% SDS), then add 10 mM DBCO-mPEG 5 kDa to a final concentration of 1 mM (1:10 dilution), and mix well.

[0075] The DBCO-mPEG-conjugated sample was incubated at 98°C for 5 min. Protein precipitation was then performed using the methanol / chloroform method: 300 μL of methanol was added to the sample and mixed; then 75 μL of chloroform and 200 μL of ultrapure water were added sequentially and mixed; centrifuged (12000 rpm, 15°C for 15 min); the supernatant was discarded, and the protein was found at the interface. Another 225 μL of methanol was added and mixed; centrifuged (12000 rpm, 15°C for 15 min), and the supernatant was discarded. The sample was air-dried at room temperature for 5 min. 20 μL of precipitation resuspension buffer B (10 mM TEA (pH 7.4), 150 mM NaCl, 1% SDS) was added to the air-dried sample to obtain the DBCO-mPEG quality-tagged enzyme-labeled O-GlcNAc glycosylated protein.

[0076] Example 3

[0077] SDS-PAGE separation and antibody-based Western blotting detection include the following steps:

[0078] 1) Sample preparation and denaturation

[0079] Take the O-GlcNAc glycosylated protein chemically labeled with the quality tag enzyme obtained in Example 2 and add 5 μL of 5× SDS loading buffer (containing DTT), and denature at 98 °C for 5 min.

[0080] 2) SDS-PAGE electrophoresis

[0081] Use a 12%–15% separating gel based on the molecular weight of α-synuclein. Mount the gel into the electrophoresis tank and add 1× electrophoresis buffer (Tris-Glycine-SDS). For sample loading: Add a protein molecular weight standard (marker) to the first well for subsequent band size determination. Add the denatured sample from step 1) to the wells. The amount added is ~40 μg, followed by SDS-PAGE electrophoresis.

[0082] 3) Transfer: Proteins were transferred to a PVDF membrane using conventional Western blotting.

[0083] 4) Blocking and antibody incubation: After the transfer, the membrane was blocked at room temperature for 1 h in TBST buffer (20 mM Tris-HCl, 500 mM sodium chloride and 0.1% Tween 20, pH 7.4) containing 5% skim milk powder, and then incubated with primary antibody: the blocked membrane was incubated overnight at 4°C with primary antibody such as α-synuclein antibody (MJFR1) or rabbit anti-O-GlcNAc.

[0084] 5) After washing and recovering the primary antibody, incubate with the secondary antibody: Incubate the washed membrane with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody at 4°C overnight or at room temperature for 2-4 hours. After incubation, wash the membrane again and recover the secondary antibody.

[0085] 6) Detection and visualization using ECL chemiluminescence: ECL chemiluminescence solution is evenly dropped onto the PVDF membrane, and the reaction is carried out in the dark for 1-2 minutes. The membrane is then placed in a chemiluminescence imaging instrument.

[0086] SDS-PAGE gel electrophoresis HPLC-MS / MS analysis includes the following steps:

[0087] Cell or tissue lysates (200 μg is preferred) were separated by SDS-PAGE electrophoresis. Proteins on the gel were stained with Coomassie Brilliant Blue R-250. After destaining, bands corresponding to the molecular weight of α-synuclein were excised against a white LED background. The excised regions or bands were determined based on the molecular weight of α-synuclein in different states in the Western blot results. These gel sections were destaining and enzymatically digested, then desalted on a C18 column, followed by vacuum freeze-drying. The resulting lyophilized powder was redissolved in 10 μL of a deionized aqueous solution containing 0.1% formic acid and centrifuged at 14000 g for 20 min. The supernatant (400 ng) was analyzed using an L-3000 HPLC system (Rigol Technologies Co. LTD.) at a flow rate of 300 nL / min. Samples were introduced into an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific Co. LTD.) equipped with a Nanospray Flex™ (NSI) ion source. Primary mass spectrometry was performed in the mass range of 350–1500 m / z with a resolution of 12000 and a maximum C-trap injection time of 50 ms. The ion spray voltage was set to 2.0 kV, and the ion transfer tube temperature was maintained at 320 °C. Secondary mass spectrometry was performed in "Extreme" mode with a resolution of 15000 (200 m / z), a maximum injection time of 22 ms, and an automatic gain control (AGC) target of 5 × 10⁻⁶. 4 MS / MS plots of modified residues were identified using Proteome Discoverer 2.4 software, with the search targets being mammalian protein databases from UniProt and NCBI.

[0088] Example 4

[0089] The quantification of O-GlcNAc-modified α-synuclein was performed using a chemical enzymatic mass spectrometry labeling strategy, as follows:

[0090] (1) Cell plating and culture

[0091] SH-SY5Y cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 °C in a 5% CO2 incubator. After washing with phosphate-buffered saline (PBS), the cells were digested with trypsin solution containing 0.25% EDTA and then re-seeded into six-well plates for overnight culture.

[0092] (2) Transfection of α-synuclein protein particles

[0093] The plasmid encoding α-synuclein was transfected using the JetPRIME® transfection reagent (PolyPlus). 2 μg of pEGFP-SNCA plasmid was dissolved in 200 μL of JetPRIME buffer, gently vortexed for 10 seconds, and then briefly centrifuged. 4 μL of JetPRIME transfection reagent was added to the diluted plasmid solution, and the mixture was vortexed to obtain a homogenate. This homogenate was incubated at room temperature for 10 min, followed by 5 min at room temperature. The mixture was then transferred to culture dishes and cultured for 36–72 h before cell harvesting.

[0094] (3) O-GlcNAc glycosylated protein catalytic coupling with GalNAz

[0095] Tissue and cell samples were homogenized in ice water with cell / tissue lysis buffer and an EDTA-free protease inhibitor. The cell / tissue lysis buffer contained 25 mM tris(hydroxymethyl)aminomethane (pH 7.5), 150 mM sodium chloride, 1% tepprofen (Triton X-100), and 20 μM PUGNAc. After incubation on ice for 30 min, the homogenized samples were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected for chemiluminescent mass spectrometry labeling. For each sample, 300 μg of protein was precipitated using methanol / chloroform extraction and then resuspended in 40 μL of protein resuspension containing 20 mM HEPES (pH 7.9) and 1% SDS. Protein concentration was determined by BCA assay, and the sample was then adjusted to 2.5 μg / μL with 20 mM HEPES buffer (pH 7.9) containing 1% SDS. The "Gal-T1 Y289L enzyme-catalyzed GalNAz coupling" method described in Example 2 was used to couple N-azidoglucose and β4GalT Y289L Enzyme-labeled the glycoproteins in the samples with terminal N-acetyl-β-D-glucosamine (GlcNAc) residues. After 20 h of labeling with N-azidoglucose, 7.5 μL of freshly prepared iodoacetamide (600 mM) was added to each sample, and the sample was then incubated in the dark by rotation for 30 min. Subsequently, the samples were precipitated using methanol / chloroform extraction, and the precipitate was dissolved in 100 μL of 10 mM triethanolamine (pH 7.4) containing 150 mM sodium chloride and 1% SDS. The amounts of reagents and reaction components used in the above steps are shown in Table 1.

[0096] Table 1. Components and concentrations of GalNAz enzyme-catalyzed ligation reaction

[0097]

[0098] Note: The solution may contain N-carbohydrate residues labeled with GalT Y289L, which can be removed by adding 1.5 μL of 500 U / μL PNGase F dissolved in 19.5 μL MilliQ-H2O.

[0099] (4) DBCO-mPEG quality label marking

[0100] To facilitate the efficient separation of α-synuclein proteins in different conformational states modified by O-GlcNAc glycosylation, 5 kDa DBCO-mPEG was used in this embodiment. 10 μL of 10 mM DBCO-mPEG (5 kDa) was added to the precipitate resuspension. After boiling at 98 °C for 5 min, the precipitate was precipitated again using methanol / chloroform extraction. Then, 40 μL of 5×SDS buffer (containing 10 mM TEA, pH 7.4; 150 mM NaCl; 1% SDS) was added to the precipitate, followed by 10 μL of 200 mM DTT solution, and the mixture was thoroughly mixed to resuspend the precipitate.

[0101] (5) SDS-PAGE separation and α-synuclein antibody recognition

[0102] Protein samples (60 μg per well) were separated by 8–12% SDS-PAGE and transferred to PVDF membranes (0.45 μm pores) using a transfer instrument. The membranes were blocked at room temperature for 1 h in TBST buffer (20 mM Tris-HCl, 500 mM sodium chloride, and 0.1% Tween 20, pH 7.4) containing 5% skim milk powder. The blocked membranes were then incubated overnight at 4 °C with primary antibodies such as rabbit anti-α-synuclein (MJFR1, ab138501, Abcam) (1:1000 dilution) or rabbit anti-O-GlcNAc (RL2, MA1-072, Thermo Scientific) (1:1000 dilution). Finally, the membranes were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (FDM007, Fdbio Science) (1:10000 dilution). Immunoreactive bands were visualized using the BioRad ECL detection system. ImageJ software (NIH) was used for band grayscale quantification, and Coomassie Brilliant Blue R-250 staining was used to confirm sample loading consistency. After catalysis by GalT Y289L enzyme, the α-synuclein expressed by the pGFR-SNCA plasmid (containing a GFP tag, molecular weight ~43 kDa) showed a higher molecular weight band (~58 kDa), indicating O-GlcNAc-modified α-synuclein. Figure 7 As shown, where Figure 7In the figure, A represents the Western blot detection results of α-synuclein O-GlcNAc modification; Figure 7 B in the figure represents the quantitative analysis of the percentage (%) of O-GlcNAc-modified α-synuclein in the total protein (~43 kDa) detected by Western blot. Thiamet-G (TMG) increases O-GlcNAc levels, promoting α-synuclein modification.

[0103] Example 5

[0104] The quantification of O-GlcNAc-modified α-synuclein was performed using an enzyme-chemical coupling combined with molecular tag labeling strategy, as follows:

[0105] (1) Expression and purification of α-synuclein

[0106] The pET21a-SNCA plasmid was introduced into BL21(DE3) *E. coli*, and expression was induced by 1 mM IPTG. The bacteria were then resuspended in a buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EGTA, and 1 mM DTT. The bacteria were sonicated, and the supernatant was passed through a Capto Q Sepharose column (GE Healthcare Life Sciences). α-synuclein was eluted with elution buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM DTT, 0.2 M NaCl). Precipitation was then performed using supersaturated ammonium sulfate. The precipitate was resuspended in 30 mM Tris-HCl (pH 7.5) and dialyzed overnight in the same buffer. After dialyzing, the solution was centrifuged at 100,000 g for 20 min at 4°C, and the supernatant was collected as the purified α-amyloid monomer. Samples were taken during the purification of recombinant α-synuclein, separated by SDS-PAGE gel electrophoresis, and stained with Coomassie brilliant blue. The results are as follows: Figure 8 As shown.

[0107] (2) Preparation of α-synuclein pre-formed fibrils (PFFs)

[0108] Purified recombinant α-synuclein protein was added to 30 mM Tris-HCl (pH=7.4) to a final concentration of 5 mg / ml. The solution was shaken counterclockwise at 1000 rpm for 5-7 days at 37 ℃. PFFs were obtained when the solution became slightly turbid. Alternatively, Western blotting was performed, using α-synuclein-specific antibodies (LB509, ab27766, Abcam) for identification, and incubation with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody (FDM007, Fdbio Science). The immunoreaction bands were visualized using a BioRad ECL detection system. The appearance of high molecular weight oligomer bands confirmed successful preparation of pre-prepared fibrils (PFFs). Recombinant α-synuclein monomers (Monomer, M) were subjected to Western blotting after 7 days of in vitro shaking. Figure 9 As shown, the PFFs group shows a high molecular weight (HMW) band, indicating that the PFFs were successfully prepared.

[0109] (3) Introduction of α-synuclein-prepared fibrils (PFFs) into cells

[0110] SH-SY5Y cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 °C in a 5% CO2 incubator. Pre-prepared fibrils (PFFs) containing α-synuclein (40 μg / mL) were used to treat SH-SY5Y cells, and cells were collected 36–72 h after transfection. PFF-treated SH-SY5Y cells were cultured for an additional 5 days.

[0111] (4) Altering cellular O-GlcNAc levels using selective inhibitors of OGA and OGT.

[0112] Cellular O-GlcNAc levels were regulated by treating cells with OGA inhibitors (Thiamet-G, TMG) ​​and OGT inhibitors (OSMI-1), and changes in α-synuclein modification were further detected. Specifically, after cells were transfected with pGFR-SNCA plasmid and cultured for 24 h, the medium was replaced with either 10 mM Thiamet-G (TMG) or 25 μM OSMI-1, and cultured for another 48 h. Western blotting was used to detect cellular O-GlcNAc glycosylation levels. The primary antibody used was rabbit anti-O-GlcNAc antibody (RL2), and the internal control was mouse anti-GAPDH antibody (FD0063, Fdbio Science). Figure 6As shown, the O-GlcNAc glycosylation level in OSMI-1 treated cells decreased compared to the control group (Ctrl), while the O-GlcNAc glycosylation level in Thiamet-G (TMG) treated cells increased.

[0113] (5) O-GlcNAc glycosylated protein catalytic coupling with GalNAz

[0114] Tissue and cell samples were placed on ice and homogenized with cell / tissue lysis buffer and an EDTA-free protease inhibitor. The cell / tissue lysis buffer contained 25 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 20 μM PUGNAc. After incubation on ice for 30 min, the supernatant was collected by centrifugation at 12,000 rpm for 15 min at 4 °C for GalNAz enzyme-catalyzed coupling. 300 μg of protein was precipitated by methanol / chloroform extraction and then resuspended in 40 μL of protein resuspension containing 20 mM HEPES (pH 7.9) and 1% SDS. Protein concentration was determined by BCA assay, and the sample was then adjusted to 2.5 μg / μL with 20 mM HEPES buffer (pH 7.9) containing 1% SDS. N-Azidose and β4GalT were coupled using the “O-GlcNAc glycosylated protein-catalyzed GalNAz coupling” method described in Example 4. Y289L Enzyme-labeled the terminal N-acetyl-β-D-glucosamine (GlcNAc) residues of the glycoprotein in the sample. After 20 h of reaction, 7.5 μL of freshly prepared iodoacetamide (600 mM) was added to the sample, and the mixture was incubated in the dark by rotation for 30 min. Subsequently, the sample was precipitated by methanol / chloroform extraction, and the precipitate was dissolved in 100 μL of 10 mM triethanolamine (pH 7.4) containing 150 mM sodium chloride and 1% SDS.

[0115] (6) DBCO-mPEG quality-tagged enzyme chemical labeling

[0116] To achieve large-scale labeling via click chemistry, 10 μL of 10 mM DBCO-mPEG (5 kDa) was added. After boiling at 98 °C for 5 min, the precipitate was extracted with methanol / chloroform. Then, 40 μL of 5×SDS buffer (10 mM TEA (pH 7.4), 150 mM sodium chloride, 1% SDS) and 10 μL of 200 mM DTT solution were added to the precipitate, and the mixture was thoroughly mixed to resuspend the precipitate.

[0117] (7) SDS-PAGE separation and Western blotting for α-synuclein antibody recognition

[0118] Protein samples (40 μg / well) were separated by 12% SDS-PAGE and transferred to PVDF membranes (0.45 μm pores) using a transfer instrument. The membranes were blocked for 1 h at room temperature in TBST buffer (20 mM Tris-HCl, 500 mM sodium chloride, and 0.1% Tween 20, pH 7.4) containing 5% skim milk powder. The blocked membranes were then incubated overnight at 4 °C with rabbit anti-O-GlcNAc (RL-2) and rabbit anti-α-synuclein (MJFR1) primary antibodies. Anti-GAPDH or anti-α-Tubulin antibodies were used as internal controls. The membranes were then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody. Immunoreactivity bands were visualized using a BioRad ECL detection system to first determine the total protein O-GlcNAc modification level. Figure 10 As shown, the O-GlcNAc level in cells treated with PFFs decreased compared to the control group, while the O-GlcNAc level increased after Thiamet-G (TMG) treatment.

[0119] Next, the O-GlcNAc-modified and unmodified α-synuclein proteins were identified based on antibody recognition. Image processing software such as ImageJ was used to quantify the grayscale values ​​of the target bands, and the ratio of O-GlcNAc-modified α-synuclein proteins was calculated. In PFF-treated cells, O-GlcNAc-modified α-synuclein proteins appeared in high molecular weight bands (~35 kDa), and were analyzed by GalT... Y289L Following enzyme catalysis, the molecular weight of O-GlcNAc-modified α-synuclein migrates upward to ~55 kDa. For example... Figure 11 As shown, where Figure 11 In the figure, A represents the Western blot detection results of α-synuclein O-GlcNAc modification; Figure 11 B in the figure represents the quantitative analysis of the percentage (%) of O-GlcNAc-modified α-synuclein in the total protein (~35 kDa) detected by Western blot. OSMI-1 treatment did not significantly affect α-synuclein modification, but Thiamet-G (TMG) increased O-GlcNAc levels and promoted α-synuclein modification.

[0120] Example 6

[0121] The O-GlcNAc site on the target α-synuclein was identified, and PFF-treated SH-SY5Y cells were homogenized and proteins extracted. Protein samples (20-60 μg per well) were denatured in a 95 ℃ metal bath for 5 min. Lysis buffer was loaded, and separation was performed using 12% SDS-PAGE. Based on a protein content of approximately ~35 kDa, the gel strips were cut into approximately 1 mm pieces using a scalpel.3 Small pieces were placed in a 1.5 mL centrifuge tube, decolorized, and shaken at a constant speed, repeated several times until decolorized and transparent. Acetonitrile was added for dehydration until the particles turned white, vacuum dried, and working solution with a final concentration of 10 mM DTT was added. The mixture was incubated at 37 ℃ for 1 h. Acetonitrile was then added for dehydration until the particles turned white, vacuum dried, and working solution with a final concentration of 55 mM IAM was added. The mixture was incubated in the dark for 30 min. Acetonitrile was added again for dehydration until the particles turned white, vacuum dried, and then washed with deionized water. This step was repeated once. 50 mM ammonium bicarbonate was added for incubation for 10 min, followed by trypsin cell digestion solution to ensure full contact between the enzyme solution and the particles. After the enzyme solution was completely absorbed by the particles, the mixture was incubated at 37 ℃ overnight. The next day, the supernatant was collected by centrifugation and placed in a new centrifuge tube. The remaining particles were added to acetonitrile and vortexed for 5 min. The supernatant was collected by centrifugation and combined with the supernatant in the previous centrifuge tube. 0.1% of the remaining particles were then added to... After rehydration, acetonitrile was added and vortexed for 5 min. The supernatant was collected and combined with the previous centrifuge tube, then lyophilized under vacuum. The sample was desalted using a C-18 desalting column. The column was activated with 100% acetonitrile, equilibrated with 0.1% formic acid, and then loaded with the sample. The column was then washed with 0.1% formic acid to remove impurities, followed by elution with 70% acetonitrile. The eluent was collected and lyophilized. The lyophilized powder was thoroughly dissolved in 0.1% formic acid solution, centrifuged at 14000 g at 4 ℃ for 20 min, and 400 ng of the supernatant was used for liquid chromatography-mass spectrometry (LC-MS). Each sample was prepared at a concentration of 10 mg / mL, with an injection volume of 25 μL.

[0122] Size exclusion chromatography (SEC) analysis was performed using a Shimadzu LC-20AD high-performance liquid chromatography system (Shimadzu, Japan), equipped with a refractive index detector (RID-20A, Shimadzu, Japan). Chromatographic separation was performed using a TSK gel G3000SWXL gel column (TOSOH, Japan) coupled with a KW-G 6B guard column. The eluent was 0.1 M sodium sulfate (pH 6.7), the flow rate was 0.5 mL / min, and the operation was carried out at 30 °C. Dual-wavelength detection was employed to enhance analytical performance; the higher wavelength (280 nm) provided a wider linear detection range, while the lower wavelength (220 nm) offered higher sensitivity for low-abundance substances. Proteomic identification was performed using mass spectrometry to identify peptides. Each precursor peptide sequence, nucleo-mass ratio (m / z) value (+203.0794), and modified residues were labeled. Figure 12-15 As shown, the mass spectrometry results indicate that the O-GlcNAc glycosylated peptide modification profile is embedded at amino acid residues S42, T44, T54, and T59.

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An in vitro detection method for α-synuclein O-GlcNAc glycosylation modification, characterized in that, Includes the following steps: (1) Extract the protein from the sample to be tested, and add UDP-GalNAz substrate and β4GalT to the protein. Y289L The mutant enzyme undergoes a reaction to obtain a protein system; (2) DBCO-mPEG is added to the protein system obtained in step (1) to carry out a copper-free click chemical reaction to obtain the reaction product; (3) The reaction products obtained in step (2) are separated by SDS-PAGE electrophoresis; if the protein in the sample to be tested has O-GlcNAc glycosylation modification, then in the electrophoresis pattern, compared with the protein without O-GlcNAc glycosylation modification, the protein with O-GlcNAc glycosylation modification shows a lag in migration rate. (4) Based on the gray values ​​of the protein bands without O-GlcNAc glycosylation and the protein bands with GlcNAc glycosylation in the electrophoresis pattern, calculate the proportion of proteins with O-GlcNAc glycosylation and their molecular weight distribution in the sample to be tested.

2. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 1, characterized in that, In step (1), the sample to be tested is selected from: cell lysate, tissue homogenate, cerebrospinal fluid or serum.

3. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 1, characterized in that, In step (2), the molecular weight of DBCO-mPEG added to the protein system is adjusted according to the polymerization state of α-synuclein in the sample to be tested.

4. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 3, characterized in that, In step (2), if the α-synuclein in the sample to be tested is a monomer, DBCO-mPEG with a molecular weight greater than or equal to 10 kDa is added to the protein system; if the α-synuclein in the sample to be tested is a polymer, DBCO-mPEG with a molecular weight of 4-6 kDa is added to the protein system.

5. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 1, characterized in that, In step (3), the reaction product is separated by SDS-PAGE electrophoresis to obtain the target protein, and the target protein is detected by immunoblotting to identify whether the protein is an α-synuclein.

6. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 1, characterized in that, In step (4), the molecular weight distribution or polymerization form of α-synuclein modified with O-GlcNAc is identified in the electrophoresis pattern based on the reference of protein molecular weight standards.

7. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 6, characterized in that, In step (4), the protein band corresponding to the α-synuclein that has undergone O-GlcNAc modification is cut and the target α-synuclein is enriched. The O-GlcNac modification site of the target α-synuclein is identified by high performance liquid chromatography-mass spectrometry.

8. The in vitro detection method for α-synuclein O-GlcNAc glycosylation modification according to claim 6, characterized in that, This method is used to screen drug candidates that regulate the level of α-synuclein O-GlcNAc modification.

9. A kit for detecting α-synuclein O-GlcNAc glycosylation modification, characterized in that, Includes: β4GalT Y289L Mutant enzyme, UDP-GalNAz substrate, and DBCO-mPEG reagent.

Images