Site complementary photoaffinity magnetic bead-target protein hooking system, preparation and application thereof

Abstract

This invention belongs to the field of screening and target identification technology for active ingredients in traditional Chinese medicine (TCM), specifically involving a site-complementary photoaffinity magnetic bead system for targeting TCM, its preparation, and application. The site-complementary photoaffinity magnetic bead combination prepared by this invention includes carboxyl-carrier-benzophenone derivative magnetic beads and amino-carrier-diacpropidine derivative magnetic beads. Both types of magnetic beads have TCM components covalently bound to their surfaces and are stably connected to photoaffinity elements via amide or ester bonds, respectively. This system can be prepared under ordinary ultraviolet light and is reusable. When used in combination, it can simultaneously capture hydrophilic / transient and hydrophobic targets, achieving more comprehensive targeting of target proteins in complex TCM systems. This overcomes the shortcomings of single magnetic beads, such as incomplete target coverage, strong equipment dependence, and easy breakage of connecting bonds. After soaking in acid or alkali, the amount of TCM components bound to the magnetic beads still maintains more than 70% of the initial value, and each 1 mg of the magnetic bead combination can target at least 0.02 μg of target protein.

Description

Technical Field

[0001] This invention belongs to the field of screening and target identification technology of active ingredients in traditional Chinese medicine, specifically involving a site-complementary photoaffinity magnetic bead system for targeting traditional Chinese medicine and its preparation and application. Background Technology

[0002] In the screening of target proteins in traditional Chinese medicine, magnetic beads with iron oxide microspheres are commonly used carrier materials. They achieve specific binding and separation of target proteins by connecting the compound or monomeric components of traditional Chinese medicine with the magnetic beads. In the existing technology, the connection method between traditional Chinese medicine components and magnetic beads has the following defects: (1) The synthesis of magnetic beads depends on special equipment, and it is necessary to make traditional Chinese medicine doped iron oxide microspheres, which is complicated; (2) Photoaffinity connection requires a professional photochemical reactor, which is expensive; (3) thiol-linked magnetic beads cannot be reused, and the hooking efficiency of target proteins is low; (4) Due to the complexity of the linkage synthesis, after binding with multiple components of traditional Chinese medicine, the magnetic beads are easily affected by protein cleaning agents, resulting in a decrease in the efficiency of magnetic beads after multiple hookings and a low concentration of hooked proteins.

[0003] To address these issues, researchers have employed various methods to prepare magnetic beads for connecting traditional Chinese medicine (TCM) using amino-carboxyl reactions. However, this method requires specific structural features, necessitating the presence of amino or carboxyl groups in the TCM components, thus limiting its applicability. Other methods utilize large-scale instruments such as Raman spectroscopy, but these are complex and expensive. Some researchers have used magnetic beads to bind to target proteins, thus targeting small TCM molecules, but this method only works for specific proteins and cannot verify the effects on multi-protein systems in animal tissues. Molecular imprinting techniques have been used to prepare magnetic beads, but their purpose is merely to enrich specific small molecules. While existing technologies involve photochemical methods for preparing TCM-linked magnetic beads, these beads often require specialized design and preparation, involving complex processes and demanding equipment. Furthermore, their subsequent use for identifying TCM target proteins requires specialized equipment such as photochemical synthesizers, placing high demands on experimental conditions.

[0004] Furthermore, considering the complexity of traditional Chinese medicine (TCM) processing and formulation, and the inherent differences in chemical composition between different production batches, target studies focusing on only a few batches may not comprehensively and reliably reflect the mechanism of action of TCM. Therefore, compared to research approaches that merely enhance the binding activity of specific components and validate them in a limited number of batches, developing microspheres of TCM compound or single-component components—characterized by simple processes, low equipment requirements, stable linkages, and reusability—can more systematically and efficiently achieve target screening and validation across multiple components and batches. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a site-complementary photoaffinity magnetic bead system for targeting traditional Chinese medicine (TCM) and its preparation and application. This invention, for the first time, constructs a site-complementary photoaffinity magnetic bead combination composed of carboxyl-carrier-benzophenone derivative magnetic beads and amino-carrier-diacrylidine derivative magnetic beads. The two types of magnetic beads are stably linked to TCM components via amide or ester bonds, respectively. Utilizing the complementary properties of benzophenone's bias towards binding hydrophobic pocket-shaped targets and diacrylidine's bias towards capturing hydrophilic and transiently acting targets, preparation and targeting can be completed under ordinary ultraviolet light irradiation. The resulting magnetic beads have stable bonds and can be reused 2-5 times. When used in combination, they can achieve more comprehensive capture of multiple components and types of target proteins in complex TCM systems, overcoming the shortcomings of existing technologies such as incomplete target coverage by single magnetic beads, strong equipment dependence, and easy breakage of bonds. This invention has significant application value and technological advancement.

[0006] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides a site-complementary photoaffinity magnetic bead assembly, comprising: a carboxyl carrier-benzophenone derivative magnetic bead and an amino carrier-diacpropidine derivative magnetic bead; wherein the surfaces of the carboxyl carrier-benzophenone derivative magnetic bead and the amino carrier-diacpropidine derivative magnetic bead are covalently bound with traditional Chinese medicine components.

[0007] Preferably, the carboxyl-carrier-benzophenone derivative magnetic beads are magnetic microspheres with carboxyl groups modified on the surface, and benzophenone photoaffinity derivatives are connected to the surface via amide or ester bonds; the amino-carrier-bisacrididine derivative magnetic beads are magnetic microspheres with amino groups modified on the surface, and bisacrididine photoaffinity derivatives are connected to the surface via amide or ester bonds.

[0008] Preferably, the particle size of the carboxyl-modified magnetic microspheres and the amino-modified magnetic microspheres are both 0.01~50 μm, and the surface functional group density is 1~1000 μmol / g; more preferably, the surface functional group density is 1~500 μmol / g; and even more preferably, the surface functional group density is 200 μmol / g.

[0009] Preferably, the benzophenone photoaffinity derivative is 4-(aminomethyl)benzophenone hydrochloride or its active ester derivative; the diacpropidine photoaffinity derivative is 3-methyl-3H-diacpropidine-3-propionic acid or its active ester derivative.

[0010] More preferably, the benzophenone photoaffinity derivative is selected from one or more of 4-(aminomethyl)benzophenone and [4-(2-amino-3-hydroxypropyl)phenyl]phenyl methyl ketone; the diacpropidine photoaffinity derivative is selected from one or more of 3-methyl-3H-diacpropidine-3-propionic acid and methyl-diacpropidine-C6-acid.

[0011] Preferably, the Chinese medicine ingredients are derived from aqueous extracts of Chinese medicine, and the aqueous extracts contain effective Chinese medicine components with methyl or methylene structures that can participate in photosynthetic reactions.

[0012] More preferably, the herbal ingredients are selected from one or more of Bupleurum chinense extract and Bupleurum chinense-Scutellaria baicalensis extract.

[0013] Preferably, after the site-complementary photoaffinity magnetic bead combination is soaked in 1 M hydrochloric acid or 1 M sodium hydroxide solution for 24 hours, the amount of traditional Chinese medicine components bound on the magnetic beads still remains above 70% of the initial value.

[0014] Preferably, the site-complementary photoaffinity magnetic bead combination can be reused 2 to 5 times, and can be reused after washing and drying with PBST buffer each time.

[0015] Preferably, each 1 mg of the site-complementary photoaffinity magnetic bead combination can hook at least 0.02 μg of target protein.

[0016] A second aspect of the present invention provides a method for preparing the site-complementary photoaffinity magnetic bead combination described in the first aspect, comprising the following steps: After activation, the carboxyl-modified magnetic microspheres react with benzophenone photoaffinity derivatives, and then covalently couple them with traditional Chinese medicine extracts under ultraviolet light irradiation to obtain carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components. After activation, amino-modified magnetic microspheres are reacted with a bisacrylidine photoaffinity derivative and then covalently coupled with a traditional Chinese medicine extract under ultraviolet light irradiation to obtain amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components. Carboxy-benzophenone magnetic beads modified with traditional Chinese medicine components and amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components were physically mixed in a certain proportion to obtain a site-complementary photoaffinity magnetic bead combination.

[0017] Preferably, in the activation step, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) is used as the condensing agent, and DMF, DMSO or other water / organic solvent miscible solvent is used as the reaction solvent. The reaction is carried out at room temperature and under stirring conditions of 100~3000 rpm for 0.5~5 hours.

[0018] Preferably, the mass ratio of the condensing agent to the carboxyl-modified magnetic microspheres is 1~50:1; the mass ratio of the condensing agent to the amino-modified magnetic microspheres is 1~50:1.

[0019] Preferably, the mass ratio of the carboxyl-modified magnetic microspheres to the traditional Chinese medicine extract is 1:10~200; the mass ratio of the amino-modified magnetic microspheres to the traditional Chinese medicine extract is 1:10~200; and the concentration of the traditional Chinese medicine extract is 10~200 mg / mL.

[0020] Preferably, the conditions for ultraviolet irradiation are: ultraviolet lamp power ≥ 10 W, wavelength 254~540 nm, irradiation distance ≤ 50 cm, and irradiation time 1~24 hours.

[0021] More preferably, the ultraviolet lamp has a power of 10~50 W and an irradiation distance of 20~50 cm.

[0022] Preferably, during the activation process, 10-500 mL of Na2CO3-NaHCO3 buffer solution may be added to adjust the pH of the reaction system to 5.0-8.0.

[0023] Preferably, the mass ratio of the carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components to the amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components is 1:10 to 10:1.

[0024] A third aspect of the present invention provides a method for target fishing of traditional Chinese medicine using a combination of site-complementary photoaffinity magnetic beads as described in the first aspect, comprising the following steps: The site-complementary photoaffinity magnetic beads combination described in the first aspect is co-incubated with a biological sample containing target proteins; the protein complex bound to the magnetic beads is collected by magnetic separation, then non-specifically bound proteins are washed away, and then the target proteins bound to the traditional Chinese medicine components are obtained by elution.

[0025] Preferably, the biological sample is a cell lysis buffer or a tissue protein extract, and the co-incubation conditions are 4°C, 20-100 rpm shaking incubation for 2-24 hours; the washing uses PBST or TBST buffer, and the washing time is 1-12 hours.

[0026] In a fourth aspect, the present invention provides a traditional Chinese medicine target hook fishing kit, comprising the site complementary photoaffinity magnetic bead combination described in the first aspect.

[0027] One or more embodiments of the present invention have at least the following beneficial effects: (1) This invention uses commercially available magnetic beads (particle size 0.05~50 μm, surface functional group density 1~1000 μmol / g) to replace the traditional self-made iron oxide microspheres doped with Chinese medicine ingredients, eliminating the need for special equipment to synthesize magnetic beads and simplifying the process; the amino and / or carboxyl groups of the magnetic beads react with the linker to form amide or ester bonds, replacing the easily oxidized thiol bonds, improving the stability of the linkage bonds and enabling the reuse of magnetic beads; commercially available three-band ultraviolet lamps (power ≥10 W, wavelength 254~365 nm) are used instead of professional photochemical reactors, along with common chemical reagents (EDC·HCl / NHS condensing agent, DMF / DMSO solvent, etc.), reducing the equipment threshold.

[0028] (2) The present invention does not require special equipment to synthesize magnetic beads. It only requires the activation and coupling of commercially available carboxyl and / or amino magnetic beads. The steps are simple and suitable for routine laboratory operations. The reaction can be completed using commercially available ordinary ultraviolet lamps (the cost is much lower than that of photochemical reactors) and ordinary quartz flasks, reducing the investment in experimental equipment.

[0029] (3) The combined structure prepared by the present invention has stronger antioxidant and acid and alkali resistance than the thiol linkage. After soaking in 1 M hydrochloric acid / sodium hydroxide for 24 hours, the amount of Chinese medicine components still retains more than 60% of the initial value, while traditional thiol magnetic beads cannot be reused due to the breakage of thiol groups. In addition, the prepared magnetic beads can be used continuously for 3-5 times. After each use, they can be reused by washing and drying with PBST buffer. When used for the 5th time, they can still effectively hook the target protein (the protein is positive after silver staining after electrophoresis separation).

[0030] (4) The magnetic bead structure prepared by the present invention has high hooking efficiency. Under the condition that sufficient protein can be bound, each 1 mg magnetic bead can hook no less than 0.2 μg of protein. Compared with traditional thiol magnetic beads, the connection bond is stable and the Chinese medicine components are covalently modified on the magnetic beads, so the binding is strong and the concentration of hooked protein is significantly increased.

[0031] (5) The magnetic bead structure obtained by the present invention has a variety of traditional Chinese medicine components: due to the complexity of traditional Chinese medicine components, multiple photoaffinity components are used, which can bind more types of traditional Chinese medicine components than a single photoaffinity component, and is more conducive to revealing the target of traditional Chinese medicine action. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 This is a flowchart of the synthesis and protein hook fishing technology of the carboxyl carrier-benzophenone derivative magnetic bead system involved in Example 1 of the present invention. In the figure, CM represents the traditional Chinese medicine extract. Figure 2 This is a flowchart of the synthesis and protein hook fishing technology of the amino-carrier-bisacrylidine derivative magnetic bead system involved in Example 2 of the present invention. In the figure, CM represents the traditional Chinese medicine extract. Figure 3 The image shows an infrared characterization comparison of benzophenone photoaffinity magnetic beads and blank magnetic beads in the carboxyl support-benzophenone derivative magnetic bead system involved in Example 1 of the present invention, where a is blank magnetic beads and b is benzophenone photoaffinity magnetic beads. Figure 4This is a comparison of infrared characterization of bisacrylidine photoaffinity magnetic beads and blank magnetic beads in the amino-carrier-bisacrylidine derivative magnetic bead system involved in Example 2 of the present invention, where a is blank magnetic beads and b is bisacrylidine photoaffinity magnetic beads; Figure 5 This is an electrophoretic image of the binding proteins of the carboxyl carrier-benzophenone derivative magnetic beads prepared in Example 1 of the present invention. In the image, a1 represents group one of protein markers; a2 represents group two of protein markers; b1 represents group one of total proteins from mouse liver tissue; b2 represents group two of total proteins from mouse liver tissue; b3 represents group three of total proteins from mouse liver tissue; c1 represents group one of benzophenone photoaffinity magnetic bead binding proteins; c2 represents group two of benzophenone photoaffinity magnetic bead binding proteins; d1 represents group one of blank magnetic bead binding proteins; d2 represents group two of blank magnetic bead binding proteins. Figure 6 This is an electrophoretic image of the binding proteins of the amino-carrier-bisacrylidine derivative magnetic beads prepared in Example 2 of the present invention. In the image, a1 represents group one of protein markers; a2 represents group two of protein markers; b1 represents group one of total proteins from mouse liver tissue; b2 represents group two of total proteins from mouse liver tissue; b3 represents group three of total proteins from mouse liver tissue; c1 represents group one of bisacrylidine photoaffinity magnetic bead binding proteins; c2 represents group two of bisacrylidine photoaffinity magnetic bead binding proteins; c3 represents group three of bisacrylidine photoaffinity magnetic bead binding proteins; d1 represents group one of blank magnetic bead binding proteins; d2 represents group two of blank magnetic bead binding proteins. Figure 7 This is an electrophoresis diagram of the binding proteins of the site-complementary photoaffinity magnetic bead combination prepared in Example 3 of the present invention. In the diagram, a1 is a group of protein markers; a2 is a group of protein markers; b1 is a group of total proteins from mouse liver tissue; b2 is a group of total proteins from mouse liver tissue; c1 is a group of site-complementary photoaffinity magnetic bead binding proteins; c2 is a group of site-complementary photoaffinity magnetic bead binding proteins; d1 is a group of blank magnetic bead binding proteins; d2 is a group of blank magnetic bead binding proteins. Detailed Implementation

[0034] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] As mentioned above, the existing technology for connecting Chinese medicine components with magnetic beads has drawbacks such as strong equipment dependence, poor stability of the bonding bonds, incomplete target capture, and limited applicability. Therefore, the present invention provides a magnetic bead system that is simple to process, has low equipment requirements, stable bonding bonds, is reusable, and can comprehensively capture multiple target points of Chinese medicine components.

[0036] A first typical embodiment of the present invention provides a site-complementary photoaffinity magnetic bead combination, comprising: a carboxyl carrier-benzophenone derivative magnetic bead and an amino carrier-diacpropidine derivative magnetic bead; both the surface of the carboxyl carrier-benzophenone derivative magnetic bead and the amino carrier-diacpropidine derivative magnetic bead are covalently bound with traditional Chinese medicine components.

[0037] In one or more embodiments of this implementation, the carboxyl support-benzophenone derivative magnetic beads are magnetic microspheres with carboxyl groups modified on the surface, and benzophenone photoaffinity derivatives are connected to the surface of the microspheres via amide bonds or ester bonds; the amino support-bisacrididine derivative magnetic beads are magnetic microspheres with amino groups modified on the surface, and bisacrididine photoaffinity derivatives are connected to the surface of the microspheres via amide bonds or ester bonds.

[0038] In one or more embodiments of this implementation, the particle size of the carboxyl-modified magnetic microspheres and the amino-modified magnetic microspheres are both 0.01~50 μm, and the surface functional group density is 1~1000 μmol / g.

[0039] In one or more embodiments of this implementation, the surface functional group density is 1~500 μmol / g.

[0040] In one or more embodiments of this implementation, the surface functional group density is 200 μmol / g.

[0041] In one or more embodiments of this implementation, the benzophenone photoaffinity derivative is 4-(aminomethyl)benzophenone hydrochloride or its active ester derivative; the diacpropidine photoaffinity derivative is 3-methyl-3H-diacpropidine-3-propionic acid or its active ester derivative.

[0042] In one or more embodiments of this implementation, the benzophenone photoaffinity derivative is selected from one or more of 4-(aminomethyl)benzophenone and [4-(2-amino-3-hydroxypropyl)phenyl]phenyl methyl ketone; the diacpropidine photoaffinity derivative is selected from one or more of 3-methyl-3H-diacpropidine-3-propionic acid and methyl-diacpropidine-C6-acid.

[0043] In one or more embodiments of this implementation, the traditional Chinese medicine ingredients are derived from aqueous extracts of traditional Chinese medicine, and the aqueous extracts of traditional Chinese medicine contain effective components of traditional Chinese medicine with methyl or methylene structures that can participate in photosynthetic reactions.

[0044] In one or more embodiments of this implementation, the traditional Chinese medicine ingredients are selected from one or more of Bupleurum chinense extract and Bupleurum chinense and Scutellaria baicalensis extract.

[0045] In one or more embodiments of this implementation, after the site-complementary photoaffinity magnetic bead combination is soaked in 1 M hydrochloric acid or 1 M sodium hydroxide solution for 24 hours, the amount of traditional Chinese medicine components bound on the magnetic beads still remains above 70% of the initial value.

[0046] In one or more embodiments of this implementation, the site-complementary photoaffinity magnetic bead combination can be reused 2 to 5 times, and can be reused after each use by washing and drying with PBST buffer.

[0047] In one or more embodiments of this implementation, each 1 mg of the site-complementary photoaffinity magnetic bead combination can hook at least 0.02 μg of target protein.

[0048] A second typical embodiment of the present invention provides a method for preparing the site-complementary photoaffinity magnetic bead combination described in the first typical embodiment, comprising the following steps: After activation, the carboxyl-modified magnetic microspheres react with benzophenone photoaffinity derivatives, and then covalently couple them with traditional Chinese medicine extracts under ultraviolet light irradiation to obtain carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components. After activation, amino-modified magnetic microspheres are reacted with a bisacrylidine photoaffinity derivative and then covalently coupled with a traditional Chinese medicine extract under ultraviolet light irradiation to obtain amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components. Carboxy-benzophenone magnetic beads modified with traditional Chinese medicine components and amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components were physically mixed in a certain proportion to obtain a site-complementary photoaffinity magnetic bead combination.

[0049] In one or more embodiments of this implementation, in the activation step, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) is used as a condensing agent, and DMF, DMSO or other water / organic solvent miscible solvent is used as the reaction solvent. The reaction is carried out at room temperature and under stirring conditions of 100~3000 rpm for 0.5~5 hours.

[0050] In one or more embodiments of this implementation, the mass ratio of the condensing agent to the carboxyl-modified magnetic microspheres is 1~50:1; the mass ratio of the condensing agent to the amino-modified magnetic microspheres is 1~50:1.

[0051] In one or more embodiments of this implementation, the mass ratio of the carboxyl-modified magnetic microspheres to the traditional Chinese medicine extract is 1:10~200; the mass ratio of the amino-modified magnetic microspheres to the traditional Chinese medicine extract is 1:10~200; and the concentration of the traditional Chinese medicine extract is 10~200 mg / mL.

[0052] In one or more embodiments of this implementation, the conditions for ultraviolet irradiation are: ultraviolet lamp power ≥10W, wavelength 254~540 nm, irradiation distance ≤50 cm, and irradiation time 1~24 hours.

[0053] In one or more embodiments of this implementation, the ultraviolet lamp is a three-band ultraviolet lamp with an irradiation wavelength of 254 nm to 365 nm, including 254 nm, 302 nm, and 365 nm. Due to the complexity of the components in traditional Chinese medicine systems, compared to single-wavelength irradiation, multi-band irradiation can obtain a wider range of more diverse target points, which can then be used for hooking and fishing various types of proteins.

[0054] In one or more embodiments of this implementation, the ultraviolet lamp has a power of 10-50 W and an irradiation distance of 20-50 cm.

[0055] In one or more embodiments of this implementation, during the activation process, 10-500 mL of Na2CO3-NaHCO3 buffer solution may be added to adjust the pH of the reaction system to 5.0-8.0.

[0056] In one or more embodiments of this implementation, the mass ratio of the carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components to the amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components is 1:10 to 10:1.

[0057] A third typical embodiment of the present invention provides a method for target fishing of traditional Chinese medicine using a combination of site-complementary photoaffinity magnetic beads as described in the first typical embodiment, comprising the following steps: The above-mentioned site-complementary photoaffinity magnetic beads were co-incubated with biological samples containing target proteins; the protein complexes bound to the magnetic beads were collected by magnetic separation, and then non-specifically bound proteins were removed by washing, followed by elution to obtain the target proteins bound to the traditional Chinese medicine components.

[0058] In one or more embodiments of this implementation, the biological sample is a cell lysis buffer or a tissue protein extract, and the co-incubation conditions are 4°C, 20-100 rpm shaking incubation for 2-24 hours; the washing uses PBST or TBST buffer, and the washing time is 1-12 hours.

[0059] A fourth typical embodiment of the present invention provides a traditional Chinese medicine target hook fishing kit, comprising the site complementary photoaffinity magnetic bead combination described in the first typical embodiment.

[0060] The protection scheme of the present invention will be described below through specific embodiments. It should be noted that these embodiments are only used to facilitate understanding by those skilled in the art and should not be regarded as limiting the scope of protection of the present invention.

[0061] In this invention, commercially available carboxyl magnetic beads were purchased from Beyotime, product number ST401-5 mL, full name BeyoMag™ Carboxyl Magnetic Beads, with a particle size of 0.2 μm and a carboxyl density of 200 μmol / g; commercially available amino magnetic beads were also purchased from Beyotime, product number P1612S-50, full name BeyoMag™ Amino Magnetic Bead Coupling Kit, with a particle size of 0.2 μm and an amino density of 200 μmol / g. Unless otherwise stated, other reagents used in the examples are commercially available.

[0062] Example 1 This embodiment provides a carboxyl-supported benzophenone derivative magnetic bead system (carboxyl-benzophenone magnetic beads linked by Bupleurum chinense extract) and its preparation method.

[0063] The synthesis of the carboxyl-carrier-benzophenone derivative magnetic bead system and the flowchart of the protein hook fishing technique are shown below. Figure 1 As shown, step 1 involves bonding carboxyl magnetic beads to benzophenone photoaffinity components to synthesize benzophenone photoaffinity magnetic beads; step 2 involves non-specific binding of benzophenone photoaffinity magnetic beads to traditional Chinese medicine components to synthesize carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components; and step 3 involves incubating the carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components with proteins for protein hook fishing.

[0064] The preparation method of the carboxyl-supported benzophenone derivative magnetic bead system specifically includes the following steps: (1) Pretreatment of magnetic beads: Take 0.01-100 g of commercial carboxyl magnetic beads with a particle size of 0.2 μm and a carboxyl density of 200 μmol / g, wash with water 2-5 times (10 mL each time), sonicate for 2 h and dry, and use immediately after treatment; (2) Activation of carboxyl magnetic beads: 100 mg of EDC·HCl was added to each 10 mg of carboxyl magnetic beads pretreated in step (1) as a condensing agent, and 10 mL of DMF was used as a solvent. The mixture was reacted with 4-(aminomethyl)benzophenone for 3 hours at room temperature and 100 rpm stirring. The unreacted raw materials were then washed 5 times by centrifugation with organic solvent DMF. The magnetic beads were then washed with water to remove the unreacted condensing agent and obtain benzophenone photoaffinity magnetic beads. (3) Coupling of Chinese medicine components with magnetic beads: 10 mL of Bupleurum chinense extract (0.1 g raw medicinal material / mL) was added to the activated magnetic beads (extraction method: 10 g of Bupleurum chinense, 100 mL of distilled water, heated and refluxed for 2 hours, extracted twice, and the extracts were combined and concentrated to 0.1 g raw medicinal material / mL). The reaction was carried out at room temperature and 100 rpm stirring for 4 hours. A commercially available three-band ultraviolet lamp with a power of 10~50W and a wavelength of 254~365 nm was used to irradiate for 300 min at an irradiation distance of 20 cm to obtain carboxyl-benzophenone magnetic beads modified with Chinese medicine components. (4) Post-processing of the product: The product was washed three times by centrifugation with PBST buffer (9000 rpm each time for 5 minutes, and the supernatant was discarded). The free Bupleurum extract components were removed by magnetic separation to obtain Bupleurum extract-linked carboxyl-benzophenone magnetic beads. The beads were stored in PBS buffer at 4°C for 30 days.

[0065] Example 2 This embodiment provides an amino-supported bisacrididine derivative magnetic bead system (amino-bisacrididine magnetic beads linked to Bupleurum chinense extract) and its preparation method.

[0066] The synthesis process of the amino-carrier-bisacrylidine derivative magnetic bead system and the protein hook fishing technique are as follows: Figure 2 As shown, step 1 involves bonding amino magnetic beads to a diazinon photoaffinity component to synthesize diazinon photoaffinity magnetic beads; step 2 involves the non-specific binding of diazinon photoaffinity magnetic beads to a traditional Chinese medicine component to synthesize amino-diazinon magnetic beads modified with the traditional Chinese medicine component; and step 3 involves using the amino-diazinon magnetic beads modified with the traditional Chinese medicine component for protein hook fishing.

[0067] The preparation method of the amino-supported bisacrylidine derivative magnetic bead system specifically includes the following steps: (1) Pretreatment of magnetic beads: Take 0.01-100 g of commercial amino magnetic beads with a particle size of 0.2 μm and an amino density of 200 μmol / g, wash with water 2-5 times (10 mL each time), sonicate for 2 h and dry, and use immediately after treatment; (2) Activation of amino magnetic beads: 100 mg of EDC·HCl was added to each 10 mg of amino magnetic beads pretreated in step (1) as a condensing agent. 20 mL of DMF was used as the reaction solvent. The mixture was reacted with 3-methyl-3H-bisacrylidine-3-propionic acid for 3 hours at room temperature and 100 rpm stirring. The unreacted raw materials were then washed 5 times by centrifugation with the organic solvent DMF. The magnetic beads were then washed with water to remove the unreacted condensing agent and obtain bisacrylidine photoaffinity magnetic beads. (3) Coupling of Chinese medicine components with magnetic beads: 10 mL of Bupleurum chinense extract (0.1 g raw medicinal material / mL) was added to the activated magnetic beads (extraction method: 10 g of Bupleurum chinense, 100 mL of distilled water, heated and refluxed for 2 hours, extracted twice, the extracts were combined and concentrated to 0.1 g raw medicinal material / mL), and reacted at room temperature and 100 rpm for 4 hours. A commercially available three-band ultraviolet lamp with a power of 10~50W and a wavelength of 254~365 nm was used to irradiate for 300 min at an irradiation distance of 20 cm to obtain amino-bisacrylidine magnetic beads modified with Chinese medicine components; (4) Post-processing of the product: The product was washed three times by centrifugation with PBST buffer (9000 rpm each time for 5 minutes, and the supernatant was discarded). The free Bupleurum extract components were removed by magnetic separation to obtain the amino-bisacrylidine magnetic beads linked to Bupleurum extract. The beads were stored in PBS buffer at 4°C for 30 days.

[0068] Example 3 This embodiment provides a traditional Chinese medicine target hook fishing system using a combination of site-complementary photoaffinity magnetic beads.

[0069] Specifically, the carboxyl-benzophenone magnetic beads linked to the Bupleurum chinense extract prepared in Example 1 and the amino-bisacrylidine magnetic beads linked to the Bupleurum chinense extract prepared in Example 2 are physically mixed in a mass ratio of 1:1 to obtain the final product.

[0070] Experimental Example 1 This experimental example characterizes the structure of the magnetic bead systems prepared in Examples 1 and 2.

[0071] like Figure 3 As shown, infrared testing revealed that at a wavenumber of 1649.36 cm⁻¹... -1 1626.62 cm -1 The presence of an amide bond (-CONH-)C=O stretching vibration peak indicates that the carboxyl magnetic beads react with the benzophenone photogroup substrate to form an amide bond, suggesting a covalent bond between the two; at a wavenumber of 1595.96 cm⁻¹. -1 1412.27 cm -1 The appearance of a benzene ring skeletal vibration peak indicates the characteristic absorption of the aromatic ring of the benzophenone core, proving that the photoreactive group has been successfully introduced.

[0072] like Figure 4 As shown, infrared testing revealed that at a wavenumber of 1622.52 cm⁻¹... -1 The presence of amide bond C=O stretching vibration at this point indicates the reaction between the carboxyl group of the diacylpropidine substrate and the amino group of the magnetic bead to form an amide bond (-CONH-). This characteristic peak suggests that the photoreactive group of diacylpropidine has been bound to the magnetic bead. The peak at wavenumber 1417.94 cm⁻¹... -1The presence of CH vibrations in the biacridine ring backbone / side chain is a characteristic absorption of the biacridine ring and alkyl side chain in the product, suggesting the introduction of photoreactive groups into the magnetic beads.

[0073] Experimental Example 1 This experimental example tests the performance of the magnetic bead systems obtained in Examples 1-3.

[0074] (1) Reusability performance test The magnetic beads prepared in Examples 1-3 were used for target protein screening. After each use, they were washed 5 times with PBS solution for 10 minutes each time, and then vacuum dried before reuse. They can be used multiple times in a row.

[0075] For Example 1, the protein content of each hook and fish was determined using the Coomassie brilliant blue staining method, and the results are as follows: Figure 5 show: In lane c, the benzophenone photoaffinity magnetic beads showed relatively obvious protein bands at 40-55 kDa and 35 kDa, while in lane d (blank magnetic beads), the bands were not obvious at the above positions.

[0076] For Example 2, the protein content of each hook and fish was determined using the Coomassie brilliant blue staining method, and the results are as follows: Figure 6 show: In lane c, the diazinon photoaffinity magnetic beads showed relatively obvious protein bands at 55-70 kDa, while in lane d (blank magnetic beads), the bands were not obvious at the same position. Furthermore, the band patterns in the three parallel lanes c1 to c3 were similar, indicating that the magnetic beads could still effectively hook proteins and produce clear staining bands even on the third use, demonstrating good reusability.

[0077] For Example 3, the protein content of each hook and fish was determined using the Coomassie brilliant blue staining method, and the results are as follows: Figure 7 show: Complementary photosynthetic magnetic beads in lane c exhibit relatively distinct protein bands at 25 kDa, 40-55 kDa, and 55-70 kDa, while lane d (blank magnetic beads) shows indistinct bands at these positions.

[0078] Compared with the single photoaffinity magnetic beads mentioned above, the site-complementary photoaffinity magnetic beads have a new protein band at 25 kDa, which indicates that the present invention may bind a richer variety of target proteins through a multi-site complementarity strategy.

[0079] (2) Connection key stability test The magnetic beads prepared in Examples 1 and 2 were immersed in 1 M hydrochloric acid or 1 M sodium hydroxide solution for 24 hours, respectively. The test results showed that the amount of traditional Chinese medicine components bound to the two types of magnetic beads remained above 70% of the initial value, indicating that the amide / ester bond linkage has excellent acid, alkali, and antioxidant capabilities.

[0080] Experimental Example 2 This experimental example tests the application of target hook-fishing proteins in the magnetic bead systems obtained in Examples 1-3.

[0081] (1) Target fishing and mass spectrometry analysis of a single magnetic bead 1) Take 200 mg of the carboxy-benzophenone magnetic beads linked to the Bupleurum chinense extract prepared in Example 1 and incubate them with 4 mL of protein extracted from the liver tissue of C57BL / 6 mice (total protein concentration 0.1~10 mg / mL, prepared using IP lysis buffer) under the following conditions: 4℃, 20 rpm shaking incubation for 2~24 hours. At the same time, take blank magnetic beads without the conjugated Chinese medicine components as a control.

[0082] 2) Remove the magnetic beads, wash them with PBST 3-5 times, discard the supernatant after magnetic separation, and wash the magnetic beads with TBST solution for 1-12 hours. The resulting washing solution is the hook and fishing protein, a traditional Chinese medicine component.

[0083] 3) Take the hook and fry protein solution, freeze dry it, and reconstitute it with PBS to obtain a sample of 0.1~1 mg / mL. Perform 10% polyacrylamide gel electrophoresis and stain with silver or Coomassie brilliant blue.

[0084] 4) Take the hook and fish protein solution, cut the protein by enzymatic digestion, desalt and concentrate it, and then use nano-liquid chromatography-mass spectrometry to detect peptides and analyze the protein. Some protein analysis results are shown in Table 1.

[0085] 5) Take 200 mg of the Bupleurum chinense extract prepared in Example 2 and link it to amino-bisacrylidine magnetic beads, and repeat steps 1) to 4). The results of some protein analysis are shown in Table 2.

[0086] (2) Target hook fishing with hybrid magnetic beads (site complementary combination) The mixed magnetic beads prepared in Example 3 were subjected to target fishing and mass spectrometry analysis following steps 1) to 4) as described above. Some protein analysis results are shown in Table 3.

[0087] Table 1. Partial protein analysis results from Example 1

[0088] Table 2. Partial protein analysis results from Example 2

[0089] Table 3. Partial protein analysis results from Example 3

[0090] Comparative analysis of the results in Tables 1-3 shows that compared to single magnetic beads, the mixed magnetic bead hooks captured significantly different and more diverse protein types, indicating that the two types of magnetic beads form effective functional complementarity at the binding sites of traditional Chinese medicine components. This invention employs a strategy combining multiple photoaffinity components, significantly expanding the coverage of proteins bound to traditional Chinese medicine. It can capture complementary target proteins that cannot be obtained by single-type magnetic beads, thereby achieving broader and more diverse target hooking in complex traditional Chinese medicine systems.

[0091] Furthermore, 10 mg of the magnetic beads prepared in Examples 1, 2, and 3 were added to a sample solution containing the target protein. After incubation at room temperature for 1 hour, the magnetic beads were collected by magnetic separation, eluted, and the amount of protein caught was determined using BSA. The results showed that each 1 mg of magnetic beads could catch 2.2 μg, 2.1 μg, and 2.1 μg of protein, respectively, meeting the requirements for target protein screening.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A site-complementary photoaffinity magnetic bead assembly, characterized in that, include: The magnetic beads are composed of a carboxyl-carrier benzophenone derivative and an amino-carrier diacylpropidine derivative. Both the carboxyl-carrier benzophenone derivative and amino-carrier diacylpropidine derivative magnetic beads have traditional Chinese medicine components covalently bonded to their surfaces. The benzophenone photoaffinity derivative is 4-(aminomethyl)benzophenone hydrochloride or its active ester derivative. The diacylpropidine photoaffinity derivative is 3-methyl-3H-diacylpropidine-3-propionic acid or its active ester derivative.

2. The site-complementary photoaffinity magnetic bead combination according to claim 1, characterized in that, The carboxyl-carrier-benzophenone derivative magnetic beads are magnetic microspheres with carboxyl groups modified on the surface, and benzophenone photoaffinity derivatives are connected to the surface of the microspheres via amide or ester bonds; the amino-carrier-bisacrididine derivative magnetic beads are magnetic microspheres with amino groups modified on the surface, and bisacrididine photoaffinity derivatives are connected to the surface of the microspheres via amide or ester bonds.

3. The site-complementary photoaffinity magnetic bead combination according to claim 2, characterized in that, The particle size of the carboxyl-modified magnetic microspheres and the amino-modified magnetic microspheres are both 0.01~50 μm, and the surface functional group density is 1~1000 μmol / g.

4. The site-complementary photoaffinity magnetic bead combination according to claim 1, characterized in that, The Chinese medicine ingredients are derived from water extracts of Chinese medicine, and the water extracts contain effective Chinese medicine components with methyl or methylene structures that can participate in photosynthetic reactions.

5. The site-complementary photoaffinity magnetic bead combination according to claim 1, characterized in that, After the site-complementary photoaffinity magnetic beads were soaked in 1 M hydrochloric acid or 1 M sodium hydroxide solution for 24 hours, the amount of traditional Chinese medicine components bound to the magnetic beads still remained above 70% of the initial value.

6. A method for preparing a site-complementary photoaffinity magnetic bead assembly according to any one of claims 1 to 5, characterized in that, Includes the following steps: After activation, the carboxyl-modified magnetic microspheres react with benzophenone photoaffinity derivatives, and then covalently couple them with traditional Chinese medicine extracts under ultraviolet light irradiation to obtain carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components. After activation, amino-modified magnetic microspheres are reacted with a bisacrylidine photoaffinity derivative and then covalently coupled with a traditional Chinese medicine extract under ultraviolet light irradiation to obtain amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components. Carboxy-benzophenone magnetic beads modified with traditional Chinese medicine components and amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components were physically mixed in a certain proportion to obtain a site-complementary photoaffinity magnetic bead combination.

7. The preparation method according to claim 6, characterized in that, In the activation step, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is used as a condensing agent, and DMF, DMSO or other water / organic solvent miscible solvent is used as the reaction solvent. The reaction is carried out at room temperature and under stirring conditions of 100~3000 rpm for 0.5~5 hours. The mass ratio of the condensing agent to the carboxyl-modified magnetic microspheres is 1~50:1; the mass ratio of the condensing agent to the amino-modified magnetic microspheres is 1~50:

1. The mass ratio of the carboxyl-modified magnetic microspheres to the traditional Chinese medicine extract is 1:10~200; the mass ratio of the amino-modified magnetic microspheres to the traditional Chinese medicine extract is 1:10~200; and the concentration of the traditional Chinese medicine extract is 10~200 mg / mL. The conditions for ultraviolet light irradiation are: ultraviolet lamp power ≥ 10 W, wavelength 254~540 nm, irradiation distance ≤ 50 cm, and irradiation time 1~24 hours; The mass ratio of the carboxyl-benzophenone magnetic beads modified with traditional Chinese medicine components to the amino-bisacrylidine magnetic beads modified with traditional Chinese medicine components is 1:10 to 10:

1.

8. A method for target fishing of traditional Chinese medicine using a combination of site-complementary photoaffinity magnetic beads as described in any one of claims 1 to 5, characterized in that, Includes the following steps: The site-complementary photoaffinity magnetic beads as described in any one of claims 1 to 5 are co-incubated with a biological sample containing target proteins; the protein complex bound to the magnetic beads is collected by magnetic separation, then non-specifically bound proteins are removed by washing, and then the target proteins bound to the traditional Chinese medicine components are obtained by elution.

9. The method according to claim 8, characterized in that, The biological sample is a cell lysis buffer or tissue protein extract. The co-incubation conditions are 4°C and shaking at 20-100 rpm for 2-24 hours. The washing is performed using PBST or TBST buffer for 1-12 hours.

10. A traditional Chinese medicine target hook fishing kit, characterized in that, It includes the site-complementary photoaffinity magnetic bead combination as described in any one of claims 1 to 5.

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