Preparation method and application of multifunctional glycoprotein photo-crosslinking agent

Abstract

The invention is applicable to the technical field of cell engineering, and provides a preparation method and application of a multifunctional glycoprotein photo-crosslinking agent, and according to the method, a biotin group type crosslinking agent containing a trifluorobis (acridine) photo-reaction active group, an oxamine sialic acid specific recognition group, a chain length-adjustable polyethylene glycol chain and a disulfide bond cleavable is prepared. The prepared multifunctional glycoprotein photo-crosslinking agent can realize efficient photo-induced protein crosslinking, and the structural design of the multifunctional glycoprotein photo-crosslinking agent is beneficial to subsequent protein decrosslinking and protein compound identification. By utilizing a trifluoro-bis-acridine photoreaction active group, the trifluoro-bis-acridine photo-crosslinking polymer has excellent photo-crosslinking activity; specific recognition and reaction on a sialic acid-containing sugar chain are realized by using an oxygen amine group; a polyethylene glycol chain with adjustable chain length is utilized to improve the solubility and biocompatibility, and cytoplasm pollution is avoided as far as possible; by utilizing biotin groups of disulfide bonds, efficient enrichment is realized, and the biotin groups can be selectively cut under a reduction condition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell engineering, and particularly relates to a preparation method and application of a multifunctional glycoprotein photo-crosslinking agent. BACKGROUND

[0002] Protein glycosylation is a widely existing and structurally complex post-translational modification of protein. Cell surface proteins are important mediators for controlling cell communication and interaction with the external environment, and the realization of the function of surface proteins largely depends on the complex glycosylation modification of surface proteins, which plays a key role in many important life processes such as cell communication, protein interaction, pathogen host recognition and the like. Analyzing the interaction network of surface glycoproteins is not only the core of revealing the rules of cell communication, but also can provide theoretical support for understanding disease mechanisms and developing new targeted drugs. However, due to the low proportion of surface proteins in mammalian whole proteins, the low abundance of surface protein glycosylation expression, and the complex structure of sugar chains, the weak / instant nature of protein glycosylation, the research on surface glycoprotein interaction still faces great challenges.

[0003] In recent years, for the research on cell surface glycoprotein, the affinity purification-mass spectrometry combined technology (AP-MS), the extracellular enzyme catalysis proximity labeling-mass spectrometry combined technology and the chemical cross-linking-mass spectrometry combined technology are mostly used. The affinity purification-mass spectrometry combined technology is the most traditional and widely used interaction mapping technology, but the affinity purification-mass spectrometry combined technology needs to analyze protein interaction in cell lysate, which not only easily destroys the weak interaction between proteins, but also affects the surface glycoprotein interaction results due to the release of cytoplasmic proteins in total cell lysate. Although the extracellular enzyme catalysis proximity labeling-mass spectrometry combined technology avoids cytoplasmic contamination, the proteins that are proximal but not directly interacting in the existing extracellular proximity labeling technology can also be labeled, and only component information can be obtained, and it is difficult to obtain specific identification information. The chemical cross-linking-mass spectrometry combined technology has developed rapidly due to its advantages such as fast analysis speed, high throughput, low requirements for protein sample amount and purity, and the ability to capture weak interactions. There are studies that combine sugar analog metabolic labeling with an azido group to the cell glycan surface, aiming to directly reveal the interaction relationship between glycosylation and other proteins in situ and systematically on the live cell membrane, but this method depends on cell culture technology and is not suitable for glycosylation labeling of tissue samples.

[0004] In addition, due to the high complexity and diversity of the structure of sugar chains, the specificity of the cross-linking agent is poor, and the spectrum analysis of the cross-linked peptide segment is difficult, resulting in low identification coverage and difficulty in obtaining structural information comprehensively. Therefore, a new type of cross-linking agent is needed to solve the above problems. SUMMARY

[0005] The purpose of the embodiments of the present application is to provide a preparation method and application of a multifunctional glycoprotein photo-crosslinking agent, which aims to solve the problems proposed in the above background technology.

[0006] The present application is implemented in a way that a preparation method of a multifunctional glycoprotein photo-crosslinking agent is provided, and the structure of the multifunctional glycoprotein photo-crosslinking agent is as follows:

[0007] The preparation method comprises the following steps: Step 1: introduction of a photo-reactive group; Boc-D-glutamic acid γ-benzyl ester α-N-hydroxysuccinimide ester (compound 1) and 4-[3-(trifluoromethyl)-3H-diazirine-3-yl] benzylamine hydrochloride are used as starting materials, anhydrous dimethyl sulfoxide (DMSO) is used as a solvent, and triethylamine (TEA) is added to carry out an amidation reaction, thereby generating (2S)-5-benzyloxy-5-oxo-2-((tert-butoxycarbonyl)amino)-N-(4-(3-(trifluoromethyl)-1H-1,2,3-triazol-4-yl)benzyl)pentanamide (compound 2) with a photoactive group.

[0008] Step 2: compound 2 is used as a reaction material, anhydrous DMSO is used as a reaction solvent, and TFA is added to carry out a reaction overnight at room temperature, thereby removing the tert-butyloxycarbonyl (Boc) protection to generate N-[4-(3-trifluoromethyl-1-pyrazolyl)benzyl]-L-2-amino-4-oxo-4-(benzyloxy)butyramide (compound 3).

[0009] Step 3: compound 3 is used as a reaction material, Pd / C is used as a catalyst, and tetrahydrofuran (THF) is used as a solvent to carry out a catalytic hydrogenation reaction under a hydrogen atmosphere at room temperature for 20 hours, thereby removing the benzyloxy protection to generate 4-amino-5-oxo-5-[(4-(3-trifluoromethyl-3H-diazirine-3-yl)benzyl)amino]pentanoic acid (compound 4).

[0010] Step 4: introduction of an enrichable and breakable group; Compound 4 and 3-[(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)disulfanyl]propanoic acid 2,5-dioxopyrrolidin-1-yl ester (NHS-SS-Biotin) are used as reaction materials, anhydrous DMSO is used as a reaction solvent, and TEA is added to carry out an amidation reaction at room temperature, thereby generating 5-oxo-4-(3-((2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)disulfanyl)propanamido)-5-((4-(3-trifluoromethyl-3H-diazirine-3-yl)benzyl)amino)pentanoic acid (compound 5) with a biotin disulfide group.

[0011] ​Step 5: introduction of a chain length-adjustable glycosyl reactive group; With compound 5 as the reaction raw material, anhydrous DMSO as the reaction solvent, 1-3-dimethylaminopropyl-3-ethyl carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) or 1-hydroxybenzotriazole (Hobt) is added to activate the carboxyl group at room temperature, and then Boc-aminooxy-polyethylene glycol-amine is added to react at room temperature to generate a polyethylene glycol chain length-adjustable tert-butyloxy ({6,11,19-trioxo-23-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9-((4-(3-trifluoromethyl-3H-diazirine-3-yl)benzyl)carbamoyl)-3,5-dioxo-14,15-dithia-4,10,18-triazatetracosyl}oxy) glycine ester (compound 6).

[0012] Step 6: With compound 6 as the reaction raw material, anhydrous DMSO as the reaction solvent, trifluoroacetic acid (TFA) is added, and the reaction is carried out at room temperature for 20 hours to remove the Boc protection to generate compound 7, which is the multifunctional glycoprotein photo-crosslinking agent.

[0013] Further technical solutions, in the step 1, the amount of 4-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzylamine hydrochloride is 0.8-1.0 molar equivalent of compound 1; Compound 1 and 4-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzylamine hydrochloride are dissolved in anhydrous DMSO, TEA is added, and the reaction is stirred at 22-30°C for 2-3 hours to obtain compound 2.

[0014] Further technical solutions, in the step 2, 0.2-0.5 mL of TFA is added to compound 2, and the reaction is stirred at room temperature overnight. The product is neutralized to slightly alkaline with ammonia water in an ice water bath, and rotary evaporation is performed. The solid after rotary evaporation is dissolved in an appropriate amount of ethyl acetate and transferred to a separatory funnel. Saturated sodium bicarbonate solution is slowly added to the separatory funnel to adjust the pH of the system to 8-9. After standing and separating the layers, the organic phase is collected; the aqueous phase is extracted with a small amount of ethyl acetate for 2-3 times, and all the organic phases are combined. The dried organic phase is rotary evaporated under reduced pressure to remove the solvent to obtain compound 3.

[0015] Further technical solutions, in the step 3, compound 3 is dissolved in THF, hydrogen is introduced, and the reaction is stirred at room temperature in the presence of Pd / C catalyst for 20 hours to obtain compound 4 after separation and purification.

[0016] Further technical solutions, in the step 4, compound 4 is dissolved in 0.8-1.2 mL of anhydrous DMSO, 3-5 molar equivalents of TEA and 1-1.2 molar equivalents of NHS-SS-Biotin are added, and then the reaction is carried out at room temperature for 4-6 hours to obtain compound 5.

[0017] Further technical solutions, in the step 5, 1.2-2 molar equivalents of EDC and 1.2-2 molar equivalents of NHS are added to compound 5, and then the activation is carried out at room temperature for 2-4 hours, after the activation is completed, Boc-aminooxy-polyethylene glycol-amine is added, and then the reaction is carried out at room temperature for 4-6 hours to obtain compound 6.

[0018] Further technical solutions, in the step 6, 10-12 molar equivalents of TFA are added to compound 6, and then the reaction is carried out at room temperature for 20 hours, and then compound 7 is obtained after separation and purification.

[0019] Another purpose of the embodiment of the present application is the application of the multifunctional glycoprotein photocrosslinking agent, the multifunctional glycoprotein photocrosslinking agent prepared based on the above method is applied to identify or analyze the interaction between the glycoprotein on the surface of living cells or tissue cells and the interacting protein.

[0020] Further technical solutions, the identification step is specifically as follows: Step a: the surface of the cells or tissue cells is subjected to oxidation treatment in advance, so as to selectively oxidize the sialic acid-containing part in the surface glycoprotein into aldehyde groups; Step b: under the condition of light, the multifunctional glycoprotein photocrosslinking agent is contacted with the oxidized cells or tissue, so that the oxime groups on the crosslinking agent react with the aldehyde groups on the glycoprotein, and at the same time, the photo-reactive groups on the crosslinking agent are crosslinked with the adjacent interacting protein under the light, so as to construct a crosslinking complex of glycoprotein-interacting protein; Step c: the constructed crosslinking complex is subjected to enrichment treatment; Step d: the sample after enrichment is analyzed by using liquid chromatography-mass spectrometry (LC-MS) and other technologies, so as to identify or analyze the glycoprotein interaction on the surface of living cells.

[0021] Through the method, the glycoprotein interaction on the surface of living cells can be comprehensively, deeply covered and accurately analyzed, and the in-situ crosslinking and identification of ligand-receptor interaction in cell communication can be used for research.

[0022] Further technical solutions, in the step a, the oxidation can be carried out by using a sodium periodate oxidation method.

[0023] The preparation method and application of the multifunctional glycoprotein photocrosslinking agent provided by the embodiment of the present application have the following beneficial effects: (1) Wide reaction range and improved coverage: By introducing trifluoro-biazinyl reactive groups, the light-activated reaction range of the cross-linking agent is wider, enabling efficient in situ cross-linking of proteins interacting with glycoproteins. This non-specific broad-spectrum cross-linking ability ensures that more potential interaction partners can be captured in a complex cellular environment, significantly improving the coverage and comprehensiveness of the cross-linking reaction, and avoiding the omission of important interaction information.

[0024] (2) Targeted precision and high specificity: By the "click chemistry" reaction of the oxime group with the pre-oxidized aldehyde group derived from sialic acid, the invention realizes highly specific targeting of cell surface glycoproteins. This design fundamentally solves the problem of poor specificity of existing technology for surface glycosylation cross-linking, accurately locks glycoproteins containing sialic acid, and greatly reduces non-specific background interference, providing high-quality, high-purity starting materials for subsequent analysis.

[0025] (3) High enrichment efficiency and good biocompatibility: The invention ingeniously introduces biotin groups with cleavable disulfide bonds into the cross-linking agent structure. Biotin endows the cross-linking agent with excellent enrichment ability, which can be efficiently separated by the streptavidin system; the presence of disulfide bonds allows specific cleavage by mild reducing conditions after enrichment, thereby efficiently releasing the cross-linked peptide segments for subsequent mass spectrometry identification, significantly improving enrichment efficiency and identification sensitivity. At the same time, this design enhances the overall biofilm targeting and biocompatibility of the cross-linking agent, reducing interference with the normal state of the cells.

[0026] (4) Excellent solubility and low background interference: By introducing polyethylene glycol chains with adjustable chain length, the invention effectively improves the solubility of the cross-linking agent in water and physiological environments, ensuring its uniform dispersion and efficient reaction in cell culture systems. The "stealth" effect of the polyethylene glycol chain not only further enhances biocompatibility, but more importantly, the steric hindrance formed by its long chain structure can effectively prevent the cross-linking agent from penetrating into the cell interior, thereby minimizing non-specific cross-linking and contamination of cytoplasmic proteins, ensuring that the analysis results are accurately focused on cell surface interactions.

[0027] In summary, the invention, through the synergistic effect of the above functional groups, has constructed an innovative technology platform that integrates broad-spectrum capture, precise targeting, efficient enrichment, biocompatibility, and low background interference. It can comprehensively, systematically, and accurately analyze the interaction network of cell surface glycoproteins, successfully overcoming the key technical bottlenecks of existing chemical cross-linking agents in glycoproteomics applications, such as poor specificity, low enrichment abundance, and high background interference. This technology provides strong theoretical support and research tools for the fields of early screening of tumors, prediction of immune therapy response, and design of antiviral drugs, and has great scientific value and application prospects. Attached Figure Description

[0028] Figure 1 Synthetic route diagram for a method of preparing a multifunctional glycoprotein photocrosslinking agent provided in an embodiment of the present invention; Figure 2 This is a cell-localization fluorescence confocal image of the chemical cross-linking agent in Example 2. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0031] like Figure 1 The image shows a method for preparing a multifunctional glycoprotein photocrosslinking agent according to an embodiment of the present invention. The structure of the multifunctional glycoprotein photocrosslinking agent is as follows: ;

[0032] Its preparation method includes the following steps: Step 1: Introduction of photoreactive groups; Using Boc-D-glutamic acid γ-benzyl ester α-N-hydroxysuccinimide ester (compound 1) and 4-[3-(trifluoromethyl)-3H-bisacrididin-3-yl]benzylamine hydrochloride as starting materials and anhydrous DMSO as solvent, TEA was added to undergo an amidation reaction to generate (2S)-5-benzyloxy-5-oxo-2-((tert-butoxycarbonyl)amino)-N-(4-(3-(trifluoromethyl)-1H-1,2,3-triazol-4-yl)benzyl)pentanamide (compound 2) with photoactive groups.

[0033] Step 2: Using compound 2 as the reactant and anhydrous DMSO as the reaction solvent, TFA was added and the reaction was carried out overnight at room temperature to remove Boc protection and generate N-[4-(3-trifluoromethyl-1-pyrazolyl)benzyl]-L-2-amino-4-oxo-4-(benzyloxy)butyramide (compound 3).

[0034] Step 3: Using compound 3 as the reactant, Pd / C as the catalyst, and THF as the solvent, the reaction was carried out at room temperature for 20 hours under a hydrogen atmosphere to undergo catalytic hydrogenation, removing benzyloxy protection and generating 4-amino-5-oxo-5-[(4-(3-trifluoromethyl-3H-diazacyclopropene-3-yl)benzyl)amino]valerate (compound 4).

[0035] Step 4: Introduction of an enrichable and cleavable group; In the presence of compound 4 and NHS-SS-Biotin as the reaction raw material, anhydrous DMSO as the reaction solvent, TEA is added, and an amidation reaction occurs at room temperature to generate 5-oxo-4-(3-((2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)disulfanyl)propanamido)-5-((4-(3-trifluoromethyl-3H-diazirine-3-yl)benzyl)amino)pentanoic acid (compound 5) with biotin disulfide.

[0036] Step 5: Introduction of a sugar-based reaction group with adjustable chain length; In the presence of compound 5 as the reaction raw material, anhydrous DMSO as the reaction solvent, EDC, NHS or Hobt is added to activate the carboxyl group at room temperature, and then Boc-aminooxy-polyethylene glycol-amine is added to react at room temperature to generate tert-butyloxy({6,11,19-trioxo-23-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9-((4-(3-trifluoromethyl-3H-diazirine-3-yl)benzyl)aminocarbonyl)-3,5-dioxo-14,15-dithia-4,10,18-triazatetracosyl}oxy)glycinate (compound 6) with adjustable polyethylene glycol chain length.

[0037] Step 6: Compound 6 as the reaction raw material, anhydrous DMSO as the reaction solvent, TFA is added, and the reaction is carried out at room temperature for 20 hours to remove the Boc protection to generate compound 7, which is the multifunctional glycoprotein photo-crosslinking agent.

[0038] As a preferred embodiment of the present application, in the step 1, the amount of 4-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzylamine hydrochloride is 0.8-1.0 mole equivalent of compound 1; Compound 1 and 4-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzylamine hydrochloride are dissolved in anhydrous DMSO, TEA is added, and the reaction is stirred at 22-30°C for 2-3 hours to obtain compound 2.

[0039] As a preferred embodiment of the present application, in the step 2, 0.2-0.5 mL of TFA is added to the compound 2, the reaction is stirred at room temperature overnight, the product is neutralized to slightly alkaline with ammonia in an ice water bath, and rotary evaporation is performed; the solid after rotary evaporation is dissolved in an appropriate amount of ethyl acetate and transferred to a separatory funnel. Saturated sodium bicarbonate solution is slowly added to the separatory funnel, and the pH of the system is adjusted to 8-9. After the layers are separated by standing, the organic phase is collected; the aqueous phase is extracted with a small amount of ethyl acetate for 2-3 times, and all the organic phases are combined. The dried organic phase is rotary evaporated under reduced pressure to remove the solvent, and the compound 3 is obtained.

[0040] As a preferred embodiment of the present application, in the step 3, the compound 3 is dissolved in THF, hydrogen is introduced, and the reaction is stirred at room temperature for 20 hours in the presence of a Pd / C catalyst, and the compound 4 is obtained after separation and purification.

[0041] As a preferred embodiment of the present application, in the step 4, the compound 4 is dissolved in 0.8-1.2 mL of anhydrous DMSO, 3-5 molar equivalents of TEA and 1-1.2 molar equivalents of NHS-SS-Biotin are added, and the reaction is performed at room temperature for 4-6 hours to obtain the compound 5.

[0042] As a preferred embodiment of the present application, in the step 5, 1.2-2 molar equivalents of EDC and 1.2-2 molar equivalents of NHS are added to the compound 5, and the reaction is activated at room temperature for 2-4 hours; after the activation is completed, Boc-aminoxy-polyethylene glycol-amine is added, and the reaction is stirred at room temperature for 4-6 hours to obtain the compound 6.

[0043] As a preferred embodiment of the present application, in the step 6, 10-12 molar equivalents of TFA are added to the compound 6, and the reaction is stirred at room temperature for 20 hours to obtain the compound 7 after separation and purification.

[0044] An application of a multifunctional glycoprotein photocrosslinking agent provided by an embodiment of the present application is based on the multifunctional glycoprotein photocrosslinking agent prepared by the above method, and the multifunctional glycoprotein photocrosslinking agent is applied to identify or analyze the interaction between a glycoprotein on the surface of a living cell or a tissue cell and an interacting protein.

[0045] The identification step is specifically as follows: Step a: the surface of a cell or a tissue cell is pre-oxidized to selectively oxidize a sialic acid-containing part in a surface glycoprotein into an aldehyde group; Step b: under light, the multifunctional glycoprotein photocrosslinking agent is contacted with the oxidized cell or tissue, so that the oxime group on the crosslinking agent reacts with the aldehyde group on the glycoprotein, and the photo-reactive group on the crosslinking agent crosslinks with the adjacent interacting protein under light, thereby constructing a glycoprotein-interacting protein crosslinking complex. Step c: enrichment treatment is performed on the constructed cross-linking complex; Step d: the sample after enrichment is analyzed by using liquid chromatography-mass spectrometry (LC-MS) and the like to identify or analyze the glycoprotein interaction on the surface of living cells.

[0046] By the method, comprehensive, deep coverage and accurate analysis of the glycoprotein interaction on the surface of living cells can be achieved, and the method can be used for in-situ cross-linking and identification of ligand-receptor interaction in cell communication.

[0047] As a preferred embodiment of the present application, in the step a, the oxidation can be performed by using sodium periodate oxidation method: After the fresh tissue slice or the whole cell is washed with pre-cooled PBS, it is immediately placed in a mixed solution of 1-2 mmol / L sodium periodate (NaIO4) and 50-100 mmol / L sodium acetate (pH = 5.5-6.0), and is treated at 4°C in the dark for 30 min to selectively oxidize the sialic acid on the surface of the cell to generate aldehyde groups; then, 10 mmol / L ethanolamine (pH = 7-8) is used for quenching at 4°C for 10 min to completely consume the residual oxidizing agent and block the newly generated aldehyde groups, and the sample is washed with PBS for 3 times, and can be directly used for subsequent photocrosslinking experiments.

[0048] The following provides several specific embodiments to verify the effectiveness of the method.

[0049] Example 1: preparation of a multifunctional glycoprotein photocrosslinking agent Step 1: compound 1 (2.17 g, 5 mmol) and 4-[3-(trifluoromethyl)-3H-diazirine-3-yl]benzylamine hydrochloride (100.69 mg, 4 mmol) are dissolved in 30 mL of anhydrous DMSO, and are stirred at 22-30°C for 2.5 hours to obtain compound 2.

[0050] Step 2: 15 mL of TFA is added to compound 2, and the reaction is stirred at room temperature overnight. The product is neutralized to slightly alkaline with ammonia water in an ice water bath, and is rotary evaporated. The solid after rotary evaporation is dissolved in an appropriate amount of ethyl acetate, and is transferred to a separatory funnel. Saturated sodium bicarbonate solution is slowly added to the separatory funnel to adjust the pH of the system to 8-9. After the layers are separated, the organic phase is collected; the aqueous phase is extracted with a small amount of ethyl acetate for 2-3 times, and all the organic phases are combined. The dried organic phase is rotary evaporated under reduced pressure to remove the solvent, and white solid product 3 is obtained.

[0051] Step 3: compound 3 is dissolved in 20 mL of THF, hydrogen is introduced, and the reaction is stirred at room temperature in the presence of Pd / C catalyst for 20 hours to obtain compound 4 after separation and purification.

[0052] Step 4: Dissolve compound 4 in 30 mL of anhydrous DMSO, add 1.52 g of TEA and 2.68 g of NHS-SS-Biotin, and react at room temperature for 4-6 hours to obtain compound 5.

[0053] Step 5: Add 1.48 g of EDC and 0.86 g of NHS to compound 5, activate at room temperature for 2-4 hours, and after activation, add Boc-aminooxy-polyethylene glycol-amine and stir at room temperature for 4-6 hours to obtain compound 6.

[0054] Step 6: Add 5.70 g of TFA to compound 6, stir and react at room temperature for 20 hours, and separate and purify to obtain compound 7, which is the multifunctional glycoprotein photocrosslinking agent (2.32 g, yield 68%).

[0055] Example 2: The multifunctional glycoprotein photocrosslinker prepared in Example 1 was applied to melanoma cells.

[0056] First, melanoma cells were gently washed with pre-chilled PBS and immediately placed in a mixed solution containing 1 mmol / L sodium periodate and 50 mmol / L sodium acetate (pH = 5.5) at 4°C in the dark for 30 minutes to selectively oxidize sialic acid on the cell surface to aldehyde groups. Then, 10 mmol / L ethanolamine (pH = 7.4) was added for quenching at 4°C for 10 minutes to completely consume residual oxidants and block newly generated aldehyde groups. The cells were then washed three times with PBS. Next, a 5 mM cross-linking agent stock solution was prepared in PBS and added to the PBS-washed melanoma cells. A chemical cross-linking reaction was performed at room temperature for 10 minutes. After the reaction, 1 mL of 10% (v / v) formaldehyde solution was added for cell fixation for 15 minutes. Then, 1 mL of 0.1% Triton X-100 solution was added to perforate the cells for 60 minutes, and the cells were incubated with 1 mL of avidin (containing fluorescein) for 1 hour. Finally, the cells were washed five times with PBS. Fluorescence confocal microscopy revealed a distinct green fluorescence on the cell surface, while no fluorescence was observed in the control group (see [link to study]). Figure 2 ).

[0057] These results demonstrate that the prepared multifunctional glycoprotein photocrosslinking agent can successfully crosslink cell surface glycoproteins without permeating the cell membrane. This experiment provides strong evidence for the large-scale analysis of cell membrane surface glycoprotein complexes and their interactions.

[0058] Example 3: The multifunctional glycoprotein photocrosslinking agent prepared in Example 1 was dissolved in PBS to prepare a crosslinking agent stock solution with a final concentration of 5 mM. This stock solution was then added to melanoma cells washed with PBS and incubated at room temperature for 10 minutes to complete the crosslinking reaction. Next, 1 mL of 8M urea and 50 mM TEAB lysis buffer were added to lyse the cells. After cell lysis, the cells were centrifuged at 12000 g, followed by protein denaturation, reduction, and alkylation. Subsequently, enzyme digestion, streptavidin magnetic bead enrichment, and desalting were performed, and finally, analysis was conducted using Lumos Fusion liquid chromatography-tandem mass spectrometry.

[0059] The obtained data were processed using pLink2 software with a false discovery rate (FDR) of 1%. Ultimately, 767 cell surface proteins and 2346 cross-linked peptides were identified, along with cross-linking information for 498 pairs of interacting glycoproteins. These results demonstrate that the prepared multifunctional glycoprotein photocrosslinking agent can achieve in-situ capture of glycoproteins on the surface of living cells and successfully perform high-coverage identification.

[0060] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multifunctional glycoprotein photocrosslinker, characterized by, The structure of the multifunctional glycoprotein photo-crosslinking agent is as follows: 。 2. A method for preparing a multifunctional glycoprotein photo-crosslinker based on the multifunctional glycoprotein photo-crosslinker according to claim 1, characterized in that, The method comprises the following steps: Step 1: introduction of a photo-reactive group; Boc-D-glutamic acid γ-benzyl ester α-N-hydroxy succinimidyl ester and 4-[3-(trifluoromethyl)-3H-diazirine-3-yl] benzylamine hydrochloride are dissolved in anhydrous DMSO, TEA is added, and stirring reaction is carried out at 22-30 DEG C for 2-3 hours to obtain compound 2. Step 2: compound 2 is used as a reaction raw material, anhydrous DMSO is used as a reaction solvent, TFA is added, and reaction is carried out at room temperature overnight to remove Boc protection and obtain compound 3. Step 3: compound 3 is used as a reaction raw material, Pd / C is used as a catalyst, and THF is used as a solvent, and catalytic hydrogenation reaction is carried out under a hydrogen atmosphere at room temperature for 20 hours to remove benzyloxy protection and obtain compound 4. Step 4: introduction of an enrichable and breakable group; Compound 4 and NHS-SS-Biotin are used as reaction raw materials, anhydrous DMSO is used as a reaction solvent, TEA is added, and amide reaction is carried out at room temperature to obtain compound 5 with biotin disulfide. Step 5: introduction of a glycosyl reaction group with adjustable chain length; Compound 5 is used as a reaction raw material, anhydrous DMSO is used as a reaction solvent, EDC, NHS or Hobt is added to activate the carboxyl group, Boc-aminoxyl-polyethylene glycol-amine is added thereto, and reaction is carried out at room temperature to obtain compound 6 containing polyethylene glycol with adjustable chain length. Step 6: compound 6 is used as a reaction raw material, anhydrous DMSO is used as a reaction solvent, TFA is added, and reaction is carried out at room temperature for 20 hours to remove Boc protection and obtain compound 7, that is, the multifunctional glycoprotein photo-crosslinking agent.

3. The preparation method of the multifunctional glycoprotein photocrosslinking agent according to claim 2, characterized in that, In the step 1, the amount of 4-[3-(trifluoromethyl)-3H-diazirine-3-yl] benzylamine hydrochloride is 0.8-1.0 mole equivalent of Boc-D-glutamic acid γ-benzyl ester α-N-hydroxy succinimidyl ester; Boc-D-glutamic acid γ-benzyl ester α-N-hydroxy succinimidyl ester and 4-[3-(trifluoromethyl)-3H-diazirine-3-yl] benzylamine hydrochloride are dissolved in anhydrous DMSO, TEA is added, and stirring reaction is carried out at 22-30 DEG C for 2-3 hours to obtain compound 2.

4. The method for preparing a multifunctional glycoprotein photocrosslinker according to claim 3, characterized by, In the step 2, 0.2-0.5 mL of TFA is added to compound 2, stirring reaction is carried out at room temperature overnight, the product is neutralized with ammonia water in an ice water bath, rotary evaporation is carried out, the solid obtained after rotary evaporation is dissolved in an appropriate amount of ethyl acetate, and then transferred to a separatory funnel; saturated sodium bicarbonate solution is slowly added to the separatory funnel, the pH of the system is adjusted to 8-9, after standing and separating, the organic phase is collected, the aqueous phase is extracted with a small amount of ethyl acetate for 2-3 times, and all the organic phases are combined; the dried organic phase is rotary evaporated under reduced pressure to remove the solvent, and compound 3 is obtained.

5. The method for preparing a multifunctional glycoprotein photocrosslinker according to claim 4, characterized by, In the step 3, compound 3 is dissolved in THF, hydrogen is introduced, and stirring reaction is carried out at room temperature in the presence of Pd / C catalyst for 20 hours to obtain compound 4 after separation and purification.

6. The method for preparing a multifunctional glycoprotein photocrosslinker according to claim 5, wherein, In the step 4, compound 4 was dissolved in 0.8-1.2 mL of anhydrous DMSO, 3-5 molar equivalents of TEA and 1-1.2 molar equivalents of NHS-SS-Biotin were added, and the reaction was carried out at room temperature for 4-6 hours to obtain compound 5.

7. The method for preparing the multifunctional glycoprotein photocrosslinking agent according to claim 6, characterized in that, In the step 5, 1.2-2 molar equivalents of EDC and 1.2-2 molar equivalents of NHS were added to compound 5, and the reaction was activated at room temperature for 2-4 hours. After the activation was completed, Boc-aminoxy-polyethylene glycol-amine was added, and the reaction was stirred at room temperature for 4-6 hours to obtain compound 6.

8. The method for preparing the multifunctional glycoprotein photocrosslinking agent according to claim 7, characterized in that, In the step 6, 10-12 molar equivalents of TFA were added to compound 6, and the reaction was stirred at room temperature for 20 hours. After separation and purification, compound 7 was obtained.

9. Use of a multifunctional glycoprotein photocrosslinker, wherein the multifunctional glycoprotein photocrosslinker is prepared according to the method of any one of claims 2 to 8, characterized in that, The multifunctional glycoprotein photo-crosslinking agent is applied to identify or analyze the interaction between the surface glycoprotein of a living cell or tissue cell and an interacting protein.

10. The use of the multifunctional glycoprotein photocrosslinker according to claim 9, characterized in that, The identification step is specifically as follows: Step a: the surface of a cell or tissue cell is pre-oxidized to selectively oxidize the sialic acid-containing part of the surface glycoprotein into an aldehyde group; Step b: under light, the multifunctional glycoprotein photo-crosslinking agent is contacted with the oxidized cell or tissue, so that the oxime group on the crosslinking agent reacts with the aldehyde group on the glycoprotein, and the photo-reactive group on the crosslinking agent crosslinks with the adjacent interacting protein under light, thereby constructing a glycoprotein-interacting protein crosslinking complex; Step c: the constructed crosslinking complex is subjected to enrichment treatment; Step d: liquid chromatography-mass spectrometry technology is used to analyze the enriched sample to identify or analyze the interaction of the surface glycoprotein of a living cell.

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