A magnetic metal-polyphenol network material based on beta-cyclodextrin and its preparation method and application

Abstract

The present invention provides a magnetic metal-polyphenol network material based on β-cyclodextrin and a preparation method and application thereof, belonging to the technical field of magnetic metal-polyphenol network material preparation method. The method obtains dopamine-derived cyclodextrin by substitution reaction; prepares magnetic nanoparticles by solvothermal method; 4+ The coordination between them forms a metal-polyphenol network on the surface of magnetic nanoparticles, i.e., the host; the reduced glutathione is modified on the adamantane to form the guest; finally, the host and the guest are mixed to obtain a magnetic metal-polyphenol network material based on β-CD; the present invention utilizes hydrophilic action to enrich glycosylated peptides, and utilizes Ti 4+ Phosphorylated peptides are enriched by coordination with phosphate ions; the paramagnetism of the internal Fe3O4 magnetic nanoparticles allows the enriched glycosylated peptides and phosphorylated peptides to be easily and quickly separated from biological samples with the assistance of external magnets, thereby improving the efficiency of the entire enrichment process.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic metal-polyphenol network material preparation methods, and specifically relates to a beta-cyclodextrin-based magnetic metal-polyphenol network material and a preparation method and application thereof. Background Art

[0002] Protein glycosylation and phosphorylation, as two important and widespread types of protein post-translational modifications, play a vital role in various biological activities. Abnormal protein glycosylation and phosphorylation are associated with diseases such as genetic diseases, neurological diseases and malignant tumors, so the detection and analysis of glycosylated peptides and phosphorylated peptides are crucial. However, due to the low abundance of glycosylated peptides and phosphorylated peptides in actual samples and their low ionization efficiency, their direct detection by mass spectrometry (MS) is hindered. Therefore, effective separation and enrichment of glycosylated peptides and phosphorylated peptides before mass spectrometry detection has become the key to the successful detection and analysis of glycosylated peptides and phosphorylated peptides. In recent years, a variety of enrichment strategies for separating glycosylated peptides and phosphorylated peptides have been constructed. Commonly used methods for enriching glycosylated peptides include hydrophilic interaction liquid chromatography, boronate affinity chromatography, hydrazine chemistry and lectin affinity chromatography; for the enrichment of phosphorylated peptides, there are methods such as immobilized metal ion affinity chromatography, metal oxide affinity chromatography and ion exchange chromatography. Here we introduced metal-polyphenol networks (MPNs) for the simultaneous enrichment of glycosylated and phosphorylated peptides.

[0003] Metal-polyphenol networks (MPNs) are metal-organic networks formed by the coordination between metal ions and phenolic ligands. They can be quickly self-assembled on substrates to form coatings, thereby achieving functional modification of substrate surfaces. MPNs are simple and rapid to prepare. Polyphenol compounds serve as organic ligands and metal ions serve as inorganic crosslinkers. Under mild conditions, the two can be quickly assembled on the target substrate to form MPNs. Due to their excellent biocompatibility, nearly universal adhesion, and mild preparation process, MPNs are widely used in biomedical fields such as bioimaging and drug delivery. The abundant hydroxyl groups and metal ions on their skeletons can enrich glycosylated peptides and phosphorylated peptides through hydrophilic interactions and fixed ion affinity, respectively. In addition, magnetic nanoparticles (MNPs) can further improve the separation efficiency due to their advantages of easy preparation, non-toxicity, good biocompatibility, and rapid magnetic responsiveness. Therefore, magnetic metal-polyphenol networks can be used to quickly separate and enrich glycosylated peptides and phosphorylated peptides.

[0004] β-cyclodextrin (β-CD) is a cyclic oligosaccharide composed of 7 D-pyranose glucose residues connected by α-1,4-glycosidic bonds. It is a macrocyclic supramolecular compound with high biocompatibility. On the one hand, the abundant hydroxyl groups on its outer surface are conducive to the enrichment of glycosylated peptides through hydrophilic interactions. On the other hand, its unique chemical structure gives it a shape-stable hydrophobic cavity, which can complex non-polar guest molecules such as adamantane (AD) through host-guest interactions. This host-guest interaction is a non-covalent, reversible, specific interaction achieved by molecular recognition. In this way, hydrophilic guests can be introduced into its hydrophobic cavity to enhance its hydrophilicity, further enhancing the affinity of the material for glycosylated peptides. Therefore, MPNs constructed based on β-CD can be non-covalently hydrophilically functionalized through host-guest interactions, thereby improving their hydrophilicity and thus enhancing their affinity for glycosylated peptides. In addition, the abundant metal ions on MPNs provide a guarantee for the enrichment of phosphorylated peptides. The synergistic effect of the two enables MPNs to be used for the simultaneous enrichment of glycosylated peptides and phosphorylated peptides in complex biological samples. Summary of the invention

[0005] The purpose of the present invention is to provide a magnetic metal-polyphenol network material based on beta-cyclodextrin and a preparation method and application thereof. The magnetic material can simultaneously separate glycosylated peptides and phosphorylated peptides from biological samples through magnetic separation.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for preparing a magnetic metal-polyphenol network material based on β-cyclodextrin comprises the following steps:

[0008] Step 1: β-CD and N,N-diimidazole are dissolved in solvents respectively, mixed and reacted in a water bath, and then dopamine hydrochloride (DA) and triethylamine (TEA) are added thereto to continue the reaction to obtain dopamine-modified β-CD (phen@β-CD);

[0009] Step 2: Dissolve 1-adamantanecarboxylic acid (AdC) in a solvent and add K 2 CO 3 , heating in a water bath, magnetic stirring, and then dropping allyl bromide, and continuing the reaction under the above conditions to obtain adamantane (Ad) containing a double bond;

[0010] Step 3: Dissolve reduced glutathione (GSH) in H 2 O and anhydrous CH 3 OH, then add it to the solution of adamantane containing double bonds obtained in step 2, add 2,2'-azobisisobutyronitrile (AIBN) in oil bath vacuum reaction to obtain GSH-modified Ad (Ad-GSH);

[0011] Step 4: Add FeCl 3 6H 2 O, NaAc and ethylene glycol (EG) were mixed and stirred evenly and then transferred to a reactor to react to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs);

[0012] Step 5: Fe obtained in step 4 3 O 4 The magnetic nanoparticles were added to the NaCl aqueous solution and ultrasonically dispersed, and then anhydrous dimethyl sulfoxide (DMSO), Ti(SO 4 ) 2 aqueous solution, anhydrous DMSO solution of dopamine-modified β-CD obtained in step 1, and an aqueous solution of 3-(N-morpholinyl)propanesulfonic acid (MOPs), heated in a water bath and stirred to react, and magnetic cyclodextrin-Ti 4+ Metal-TiCD network materials (mTiCD);

[0013] Step 6: Disperse the GSH-modified Ad obtained in step 3 in a solvent, and add the magnetic cyclodextrin-Ti prepared in step 5 4+ The metal-polyphenol network material aqueous solution is ultrasonically dispersed and then transferred to a water bath, heated and stirred for reaction to obtain a β-CD-based magnetic metal-polyphenol network material (mTiCD@AG).

[0014] Preferably, in the step 1, the mass ratio of β-CD, N,N′-carbonyldiimidazole and dopamine hydrochloride is 2.28:1.62:1.9.

[0015] Preferably, in the step 2, 1-carboxyadamantane and K 2 CO 3 The mass ratio is 5.4:5.

[0016] Preferably, in step 3, the vacuum reaction temperature is 60-63° C. and the reaction time is 22-24 h.

[0017] Preferably, the reaction temperature in step 4 is 180-200° C. and the reaction time is 14-16 h.

[0018] Preferably, in step 5, MNPs mg: NaCl aqueous solution mL: anhydrous dimethyl sulfoxide mL: Ti(SO 4 ) 2Aqueous solution mL: phen@CD anhydrous dimethyl sulfoxide solution mL: 3-(N-morpholino)propanesulfonic acid mL is (30-90): (2.3-6.9): (0.48-1.44): (0.18-0.54): (0.18-0.54): (3-9).

[0019] Preferably, the reaction temperature of step six is ​​21-23° C., and the reaction time is 2-4 h.

[0020] The present invention also provides a magnetic metal-polyphenol network material based on β-CD obtained by the above preparation method.

[0021] Preferably, the β-CD-based magnetic metal-polyphenol network material is spherical in appearance, with a size concentrated in the range of 230-260 nm and paramagnetic.

[0022] The present invention also provides the use of the above-mentioned magnetic metal-polyphenol network material based on β-CD in the separate and simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides.

[0023] Beneficial effects of the present invention

[0024] The present invention provides a magnetic metal-polyphenol network material based on β-CD and a preparation method and application thereof. The method comprises the following steps: substituting and modifying β-cyclodextrin with dopamine hydrochloride to obtain dopamine-derived cyclodextrin; and preparing Fe by a solvothermal method. 3 O 4 Magnetic nanoparticles; and then using Ti 4+ The metal-polyphenol network is covered on the surface of the magnetic sphere by the coordination between the ortho-hydroxyl groups on dopamine, and the obtained magnetic metal-polyphenol network is the main body; the diamond is modified by GSH to obtain the guest molecule; finally, the main magnetic metal-polyphenol network and the guest GSH-modified diamond are mixed to obtain the magnetic metal-polyphenol network material based on β-CD through the host-guest interaction; the present invention utilizes the hydrophilicity of the hydroxyl groups on the surface of β-CD and the carboxyl groups on GSH, so that the hydrophilic glycosylated peptide modified with polysaccharide is enriched on the surface of the material, and the rich Ti in the metal-polyphenol network 4+ It can coordinate with phosphate, so that phosphorylated peptides are enriched on the surface of the material; while the internal Fe 3 O 4 The paramagnetism of magnetic nanoparticles allows the enriched glycosylated peptides and phosphorylated peptides to be easily and quickly separated from biological samples with the assistance of an external magnet, making it more convenient to detect and analyze glycosylated peptides and phosphorylated peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a SEM image of the MNPs prepared in Example 1 of the present invention;

[0026] Figure 2 This is a SEM image of the mTiCD prepared in Example 1 of the present invention;

[0027] Figure 3 This is a SEM image of mTiCD@AG prepared in Example 1 of the present invention;

[0028] Figure 4 hysteresis loop diagrams of three magnetic materials prepared in Example 1 of the present invention;

[0029] Figure 5 This is the mass spectrum of the mTiCD@AG prepared in Example 1 of the present invention that enriches glycosylated peptides alone.

[0030] Figure 6 This is the mass spectrum of the mTiCD@AG prepared in Example 1 of the present invention that enriches phosphorylated peptides alone.

[0031] Figure 7 This is the mass spectrum of mTiCD@AG prepared in Example 1 of the present invention that simultaneously enriches glycosylated peptides and phosphorylated peptides. Specific implementation plan

[0032] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0033] Step 1: β-CD and N,N′-carbonyldiimidazole are dissolved in a solvent, preferably anhydrous N,N-dimethylformamide, and the two solutions are mixed after being fully dissolved, preferably reacting in a water bath at 20-23° C. for 12-13 hours. Then, dopamine hydrochloride (DA) and triethylamine (TEA) are added to the reaction solution in sequence, and the reaction is preferably continued in a water bath for 15-16 hours. After the reaction is completed, the obtained solution is added dropwise to acetone, allowed to stand at room temperature, and then centrifuged (10000 rpm, 5 min). The standing time is preferably 1 h, and the white precipitate is separated. The white precipitate is then dispersed again with acetone, and then centrifuged under the same conditions. The obtained product is vacuum dried to obtain dopamine-modified β-CD (phen@CD); the vacuum drying temperature is preferably 50°C, and the time is 16 h; the mass ratio of β-CD, N,N′-carbonyldiimidazole and dopamine hydrochloride is preferably 2.28:1.62:1.9; the mass g of dopamine hydrochloride: the volume mL of triethylamine is preferably 1.9:2.8;

[0034] Step 2: Dissolve 1-carboxyadamantane in a solvent, preferably anhydrous dimethyl sulfoxide, and add K 2 CO 3 , preferably under 20-23°C water bath magnetic stirring for 22-24h, after the reaction is completed, the reaction product is filtered, the precipitate is discarded and the filtrate is retained. 滤液:V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V 乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层 :V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4 The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator. The 1-carboxyadamantane and K 2 CO 3 The mass ratio is preferably 5.4:5;

[0035] Step 3: Dissolve the double-bonded adamantane in H 2 O and anhydrous methanol, the mixture contains H 2 The volume ratio of O and anhydrous methanol is preferably 2:3; GSH (reduced glutathione) is dissolved in H 2 O and anhydrous methanol in a mixture; H 2 The volume ratio of O and anhydrous methanol is preferably 3:2; the above two solutions are mixed, and azobisisobutyronitrile is added, and N 2 The reaction is carried out in vacuum, preferably at 60-63°C with oil bath magnetic stirring for 22-24h. After the reaction is completed, the reaction solution is cooled to room temperature, vacuum rotary evaporation is performed to obtain a solid, and the obtained product is vacuum dried to obtain adamantane modified with GSH (Ad-GSH), which is placed in a refrigerator for storage; the vacuum drying temperature is preferably 40°C, and the time is preferably 16h; the mass ratio of the product adamantane modified with double bonds, GSH and azobisisobutyronitrile in step 2 is preferably 1:1:0.05;

[0036] Step 4: Add FeCl 3 6H 2 O and NaAc are dissolved in ethylene glycol, mixed with magnetic stirring, the stirring time is preferably 1-2h, transferred to a high temperature reactor, preferably reacted at 180-200°C for 14-16h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and dried under vacuum to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs); the FeCl 3 6H 2The mass ratio of O and NaAc is preferably 1.35:3.6;

[0037] Step 5: Add MNPs to NaCl aqueous solution, add anhydrous dimethyl sulfoxide after ultrasonic dispersion, and then add Ti(SO 4 ) 2 Then, phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution, and the mixture was transferred to a water bath preferably at a temperature of 21-23°C, and 3-(N-morpholinyl)propanesulfonic acid was added. The mechanical stirring time under the above conditions was preferably 2-4h, and H 2 O and ethanol twice respectively, and vacuum dried to obtain a magnetic metal-polyphenol network (mTiCD); the vacuum drying temperature is preferably 40-50°C, the time is preferably 14-16h, the concentration of the NaCl aqueous solution is preferably 0.5M, Ti(SO 4 ) 2 The concentration of the aqueous solution is preferably 100 mM, the concentration of the phen@CD anhydrous dimethyl sulfoxide solution is preferably 100 mM, the concentration of 3-(N-morpholinyl)propanesulfonic acid is preferably 20 mM, and the mass mg of the MNPs: the volume mL of the NaCl aqueous solution is preferably (30-90): (2.3-6.9); the NaCl aqueous solution, anhydrous dimethyl sulfoxide, Ti(SO 4 ) 2 The volume ratio of the aqueous solution, the phen@CD anhydrous dimethyl sulfoxide solution and 3-(N-morpholino)propanesulfonic acid is preferably (2.3-6.9):(0.48-1.44):(0.18-0.54):(0.18-0.54):(3-9).

[0038] Step 6: Disperse the Ad-GSH obtained in step 3 in a solvent, preferably anhydrous dimethyl sulfoxide, and dissolve the mTiCD prepared in step 5 in H 2 O, and then the two solutions are mixed and ultrasonicated, the ultrasonic time is preferably 9-12 minutes, and then the mixed solution is placed in a 21-23°C water bath and stirred for 2-4 hours, washed once with dimethyl sulfoxide, washed twice with ethanol, and vacuum dried to obtain a β-CD-based magnetic metal-polyphenol network material mTiCD@AG, the vacuum drying temperature is preferably 40-50°C, the time is preferably 14-16 hours, and the mass ratio of Ad-GSH to mTiCD is preferably 2:1.

[0039] The present invention also provides a magnetic metal-polyphenol network material based on β-CD obtained by the above preparation method. The magnetic metal-polyphenol network material is spherical in appearance, with a size concentrated in 230-260nm and has strong paramagnetism.

[0040] The present invention also provides the application of the above-mentioned magnetic metal-polyphenol network material based on β-cyclodextrin in separating and enriching proteins, and the specific steps are as follows:

[0041] (1) Separation and enrichment of glycosylated peptides

[0042] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0043] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0044] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2 The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0045] (2) Separation and enrichment of phosphorylated peptides

[0046] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0047] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0048] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H 2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0049] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0050] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0051] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0052] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0053] The present invention is described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0054] Unless otherwise defined, the terms (including technical terms) used herein should be interpreted as having the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It will also be understood that the terms used herein should be interpreted as having the meaning consistent with their meanings in the content of this specification and related technologies, and should not be interpreted in an idealized or excessive form unless specifically defined as such herein.

[0055] Example 1

[0056] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0057] (1) 2.28 g of β-CD and 1.62 g of N,N′-carbonyldiimidazole were dissolved in 10 mL of anhydrous N,N-dimethylformamide, and the two solutions were mixed after being fully dissolved, and reacted in a water bath at 23°C for 12 h. Then, 1.9 g of dopamine hydrochloride and 2.8 mL of triethylamine were added to the reaction solution in sequence, and the reaction was continued in a water bath at 23°C for 16 h. After the reaction was completed, the obtained solution was added dropwise to 400 mL of acetone, allowed to stand at room temperature for 1 h, and then centrifuged (10000 rpm, 5 min) to separate the white precipitate, and then the white precipitate was dispersed again with acetone, and then centrifuged under the same conditions. The obtained product was vacuum dried at 50°C for 16 h to obtain dopamine-modified β-CD (phen@CD).

[0058] (2) Dissolve 5.4 g of 1-carboxyadamantane in 150 mL of anhydrous dimethyl sulfoxide and add 5 g of K 2 CO 3, stirred in a water bath with magnetic stirring at 23°C for 24 hours, after the reaction, the reaction product was filtered, the precipitate was discarded and the filtrate was retained. 滤液 :V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V 乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层 :V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4 The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator.

[0059] (3) Dissolve 1 g of the product modified with double bonds in 10 mL of H 2 In a mixture of 1 mL of H O and 15 mL of anhydrous methanol, 1 g of GSH was dissolved in 3 mL of H 2 O and 2mL of anhydrous methanol; mix the above two solutions, add 50mg of azobisisobutyronitrile, and pass N 2 The reaction was carried out in a vacuum, and the mixture was stirred in an oil bath under magnetic stirring at 60° C. for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, and a solid was obtained by vacuum rotary evaporation. The obtained product was vacuum dried at 40° C. for 16 h to obtain adamantane modified with GSH (Ad-GSH), which was stored in a refrigerator.

[0060] (4) 1.35 g FeCl 3 6H 2 O, 3.6 g NaAc was dissolved in 25 mL ethylene glycol, mixed and magnetically stirred for 1 h, transferred to a high-temperature reactor, reacted at 200 ° C for 16 h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and vacuum dried at 50 ° C to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs); SEM images are shown in Figure 1 shown.

[0061] (5) 30 mg of MNPs were added to 2.3 mL of 0.5 M NaCl aqueous solution, and after ultrasonic dispersion, 480 μL of anhydrous dimethyl sulfoxide was added, followed by 180 μL of 100 mM Ti (SO 4 ) 2Then, 180 μL of 100 mM phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution. The mixture was transferred to a 23°C water bath, and 3 mL of 20 mM 3-(N-morpholinyl)propanesulfonic acid was added. The mixture was mechanically stirred for 2 h under the above conditions, and then heated with H 2 O and ethanol were washed twice, and vacuum dried at 50℃ for 16h to obtain magnetic metal-polyphenol network (mTiCD). Figure 2 shown.

[0062] (6) Disperse 30 mg Ad-GSH in 50 mL anhydrous dimethyl sulfoxide and dissolve 15 mg mTiCD in 10 mL H 2 O, and then the two solutions were mixed and ultrasonicated for 10 min. The mixture was then placed in a 23 °C water bath and stirred for 2 h. The mixture was washed once with dimethyl sulfoxide and twice with ethanol. The mixture was vacuum dried at 40 °C for 16 h to obtain the β-CD-based magnetic metal-polyphenol network material mTiCD@AG.

[0063] Figure 3 This is the SEM image of mTiCD@AG obtained in the present invention. Figure 3 It shows that mTiCD@AG is spherical in appearance and its size is concentrated in the range of 230-260nm.

[0064] Figure 4 The hysteresis loop diagrams of MNP, mTiCD and mTiCD@AG obtained in the present invention indicate that the prepared magnetic materials have certain paramagnetism.

[0065] Example 1 The application of magnetic materials in separating and enriching glycoproteins specifically includes:

[0066] (1) Separation and enrichment of glycosylated peptides

[0067] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0068] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0069] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0070] (2) Separation and enrichment of phosphorylated peptides

[0071] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0072] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0073] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H 2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0074] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0075] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2 O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0076] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0077] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0078] like Figure 5-7 As shown, Figure 5 This is the mass spectrum of the mTiCD@AG prepared in Example 1 of the present invention that enriches glycosylated peptides alone. Figure 6This is the mass spectrum of the mTiCD@AG prepared in Example 1 of the present invention that enriches phosphorylated peptides alone. Figure 7 This is the mass spectrum of mTiCD@AG prepared in Example 1 of the present invention that simultaneously enriches glycosylated peptides and phosphorylated peptides. Figure 5-7 This indicates that mTiCD@AG has a certain ability to enrich glycosylated peptides and phosphorylated peptides.

[0079] Example 2

[0080] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0081] (1) 2.28 g of β-CD and 1.62 g of N,N′-carbonyldiimidazole were dissolved in 10 mL of anhydrous N,N-dimethylformamide, and the two solutions were mixed after being fully dissolved, and reacted in a water bath at 23°C for 12 h. Then, 1.9 g of dopamine hydrochloride and 2.8 mL of triethylamine were added to the reaction solution in sequence, and the reaction was continued in a water bath at 23°C for 16 h. After the reaction was completed, the obtained solution was added dropwise to 400 mL of acetone, allowed to stand at room temperature for 1 h, and then centrifuged (10000 rpm, 5 min) to separate the white precipitate, and then the white precipitate was dispersed again with acetone, and then centrifuged under the same conditions. The obtained product was vacuum dried at 50°C for 16 h to obtain dopamine-modified β-CD (phen@CD).

[0082] (2) Dissolve 5.4 g of 1-carboxyadamantane in 150 mL of anhydrous dimethyl sulfoxide and add 5 g of K 2 CO 3 , stirred in a water bath with magnetic stirring at 23°C for 24 hours, after the reaction, the reaction product was filtered, the precipitate was discarded and the filtrate was retained. 滤液 :V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V 乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层 :V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4 The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator.

[0083] (3) Dissolve 1 g of the product modified with double bonds in 10 mL of H 2 In a mixture of 1 mL of H O and 15 mL of anhydrous methanol, 1 g of GSH was dissolved in 3 mL of H 2 O and 2mL of anhydrous methanol; mix the above two solutions, add 50mg of azobisisobutyronitrile, and pass N 2 The reaction was carried out in a vacuum, and the mixture was stirred in an oil bath under magnetic stirring at 60° C. for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, and a solid was obtained by vacuum rotary evaporation. The obtained product was vacuum dried at 40° C. for 16 h to obtain adamantane modified with GSH (Ad-GSH), which was stored in a refrigerator.

[0084] (4) 2.43 g FeCl 3 6H 2 O, 6.48g NaAc was dissolved in 45mL ethylene glycol, mixed and magnetically stirred for 1h, transferred to a high-temperature reactor, reacted at 200℃ for 16h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and dried in vacuum at 50℃ to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs).

[0085] (5) 30 mg of MNPs were added to 2.3 mL of 0.5 M NaCl aqueous solution, and after ultrasonic dispersion, 480 μL of anhydrous dimethyl sulfoxide was added, followed by 180 μL of 100 mM Ti (SO 4 ) 2 Then, 180 μL of 100 mM phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution. The mixture was transferred to a 23°C water bath, and 3 mL of 20 mM 3-(N-morpholinyl)propanesulfonic acid was added. The mixture was mechanically stirred for 2 h under the above conditions, and then heated with H 2 The resulting product was washed twice with O and ethanol respectively and dried in vacuum at 50 °C for 16 h to obtain the magnetic metal-polyphenol network (mTiCD).

[0086] (6) Disperse 30 mg Ad-GSH in 50 mL anhydrous dimethyl sulfoxide and dissolve 15 mg mTiCD in 10 mL H 2 O, and then the two solutions were mixed and ultrasonicated for 10 min. The mixture was then stirred in a 23 °C water bath for 3 h, washed once with dimethyl sulfoxide and twice with ethanol, and vacuum dried at 40 °C for 16 h to obtain the magnetic metal-polyphenol network material mTiCD@AG based on the host-guest interaction between β-CD and adamantane.

[0087] Example 2 The application of magnetic materials in separating and enriching proteins specifically includes:

[0088] (1) Separation and enrichment of glycosylated peptides

[0089] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0090] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0091] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2 The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0092] (2) Separation and enrichment of phosphorylated peptides

[0093] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0094] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0095] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H 2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0096] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0097] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2 O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0098] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0099] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0100] Example 3

[0101] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0102] (1) 2.28 g of β-CD and 1.62 g of N,N′-carbonyldiimidazole were dissolved in 10 mL of anhydrous N,N-dimethylformamide, and the two solutions were mixed after being fully dissolved, and reacted in a water bath at 23°C for 12 h. Then, 1.9 g of dopamine hydrochloride and 2.8 mL of triethylamine were added to the reaction solution in sequence, and the reaction was continued in a water bath at 23°C for 16 h. After the reaction was completed, the obtained solution was added dropwise to 400 mL of acetone, allowed to stand at room temperature for 1 h, and then centrifuged (10000 rpm, 5 min) to separate the white precipitate, and then the white precipitate was dispersed again with acetone, and then centrifuged under the same conditions. The obtained product was vacuum dried at 50°C for 16 h to obtain dopamine-modified β-CD (phen@CD).

[0103] (2) Dissolve 5.4 g of 1-carboxyadamantane in 150 mL of anhydrous dimethyl sulfoxide and add 5 g of K 2 CO 3 , stirred in a water bath with magnetic stirring at 23°C for 24 hours, after the reaction, the reaction product was filtered, the precipitate was discarded and the filtrate was retained. 滤液 :V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V 乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层 :V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator.

[0104] (3) Dissolve 1 g of the product modified with double bonds in 10 mL of H 2 In a mixture of 1 mL of H O and 15 mL of anhydrous methanol, 1 g of GSH was dissolved in 3 mL of H 2 O and 2mL of anhydrous methanol; mix the above two solutions, add 50mg of azobisisobutyronitrile, and pass N 2 The reaction was carried out in a vacuum, and the mixture was stirred in an oil bath under magnetic stirring at 60° C. for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, and a solid was obtained by vacuum rotary evaporation. The obtained product was vacuum dried at 40° C. for 16 h to obtain adamantane modified with GSH (Ad-GSH), which was stored in a refrigerator.

[0105] (4) 2.7 g FeCl 3 6H 2 O, 7.2g NaAc was dissolved in 50mL ethylene glycol, mixed and magnetically stirred for 1h, transferred to a high-temperature reactor, reacted at 200℃ for 16h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and dried in vacuum at 50℃ to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs).

[0106] (5) 60 mg of MNPs were added to 4.6 mL of 0.5 M NaCl aqueous solution, and after ultrasonic dispersion, 0.96 mL of anhydrous dimethyl sulfoxide was added, followed by 0.36 mL of 100 mM Ti(SO 4 ) 2 Then, 0.36 mL of 100 mM phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution. The mixture was transferred to a 23°C water bath, and 6 mL of 20 mM 3-(N-morpholinyl)propanesulfonic acid was added. The mixture was mechanically stirred for 3 h under the above conditions, and then heated with H 2 The resulting product was washed twice with O and ethanol respectively and dried in vacuum at 50 °C for 16 h to obtain the magnetic metal-polyphenol network (mTiCD).

[0107] (6) Disperse 30 mg Ad-GSH in 50 mL anhydrous dimethyl sulfoxide and dissolve 15 mg mTiCD in 10 mL H 2 O, and then the two solutions were mixed and ultrasonicated for 10 min. The mixture was then stirred in a 23 °C water bath for 3 h, washed once with dimethyl sulfoxide and twice with ethanol, and vacuum dried at 40 °C for 16 h to obtain the magnetic metal-polyphenol network material mTiCD@AG based on the host-guest interaction between β-CD and adamantane.

[0108] Example 3 Application of magnetic materials in separating and enriching proteins, specifically including:

[0109] (1) Separation and enrichment of glycosylated peptides

[0110] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0111] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0112] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2 The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0113] (2) Separation and enrichment of phosphorylated peptides

[0114] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0115] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0116] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H 2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0117] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0118] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0119] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0120] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0121] Example 4

[0122] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0123] (1) 2.28 g of β-CD and 1.62 g of N,N′-carbonyldiimidazole were dissolved in 10 mL of anhydrous N,N-dimethylformamide, and the two solutions were mixed after being fully dissolved, and reacted in a water bath at 23°C for 12 h. Then, 1.9 g of dopamine hydrochloride and 2.8 mL of triethylamine were added to the reaction solution in sequence, and the reaction was continued in a water bath at 23°C for 16 h. After the reaction was completed, the obtained solution was added dropwise to 400 mL of acetone, allowed to stand at room temperature for 1 h, and then centrifuged (10000 rpm, 5 min) to separate the white precipitate, and then the white precipitate was dispersed again with acetone, and then centrifuged under the same conditions. The obtained product was vacuum dried at 50°C for 16 h to obtain dopamine-modified β-CD (phen@CD).

[0124] (2) Dissolve 5.4 g of 1-carboxyadamantane in 150 mL of anhydrous dimethyl sulfoxide and add 5 g of K 2 CO 3 , stirred in a water bath with magnetic stirring at 23°C for 24 hours, after the reaction, the reaction product was filtered, the precipitate was discarded and the filtrate was retained. 滤液 :V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V 乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层:V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4 The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator.

[0125] (3) Dissolve 1 g of the product modified with double bonds in 10 mL of H 2 In a mixture of 1 mL of H O and 15 mL of anhydrous methanol, 1 g of GSH was dissolved in 3 mL of H 2 O and 2mL of anhydrous methanol; mix the above two solutions, add 50mg of azobisisobutyronitrile, and pass N 2 The reaction was carried out in a vacuum, and the mixture was stirred in an oil bath under magnetic stirring at 60° C. for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, and a solid was obtained by vacuum rotary evaporation. The obtained product was vacuum dried at 40° C. for 16 h to obtain adamantane modified with GSH (Ad-GSH), which was stored in a refrigerator.

[0126] (4) 3gFeCl 3 6H 2 O, 8g NaAc was dissolved in 56mL ethylene glycol, mixed and magnetically stirred for 1h, transferred to a high-temperature reactor, reacted at 200℃ for 16h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and dried in vacuum at 50℃ to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs).

[0127] (5) 90 mg of MNPs were added to 6.9 mL of 0.5 M NaCl aqueous solution, and after ultrasonic dispersion, 1.44 mL of anhydrous dimethyl sulfoxide was added, followed by 0.54 mL of 100 mM Ti(SO 4 ) 2 Then, 0.54 mL of 100 mM phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution. The mixture was transferred to a 23°C water bath, and 9 mL of 20 mM 3-(N-morpholinyl)propanesulfonic acid was added. The mixture was mechanically stirred for 4 h under the above conditions, and then heated with H 2 The resulting product was washed twice with O and ethanol respectively and dried in vacuum at 50 °C for 16 h to obtain the magnetic metal-polyphenol network (mTiCD).

[0128] (6) Disperse 30 mg Ad-GSH in 50 mL anhydrous dimethyl sulfoxide and dissolve 15 mg mTiCD in 10 mL H 2O, and then the two solutions were mixed and ultrasonicated for 10 min. The mixture was then placed in a 23 °C water bath and stirred for 2 h. The mixture was washed once with dimethyl sulfoxide and twice with ethanol. The mixture was vacuum dried at 40 °C for 16 h to obtain the magnetic metal-polyphenol network material mTiCD@AG based on the host-guest interaction between β-CD and adamantane.

[0129] Example 4 The application of magnetic materials in separating and enriching proteins specifically includes:

[0130] (1) Separation and enrichment of glycosylated peptides

[0131] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0132] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0133] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2 The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0134] (2) Separation and enrichment of phosphorylated peptides

[0135] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0136] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0137] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H 2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0138] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0139] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2 O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0140] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0141] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0142] Example 5

[0143] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0144] (1) 2.28 g of β-CD and 1.62 g of N,N′-carbonyldiimidazole were dissolved in 10 mL of anhydrous N,N-dimethylformamide, and the two solutions were mixed after being fully dissolved, and reacted in a water bath at 23°C for 12 h. Then, 1.9 g of dopamine hydrochloride and 2.8 mL of triethylamine were added to the reaction solution in sequence, and the reaction was continued in a water bath at 23°C for 16 h. After the reaction was completed, the obtained solution was added dropwise to 400 mL of acetone, allowed to stand at room temperature for 1 h, and then centrifuged (10000 rpm, 5 min) to separate the white precipitate, and then the white precipitate was dispersed again with acetone, and then centrifuged under the same conditions. The obtained product was vacuum dried at 50°C for 16 h to obtain dopamine-modified β-CD (phen@CD).

[0145] (2) Dissolve 5.4 g of 1-carboxyadamantane in 150 mL of anhydrous dimethyl sulfoxide and add 5 g of K 2 CO 3 , stirred in a water bath with magnetic stirring at 23°C for 24 hours, after the reaction, the reaction product was filtered, the precipitate was discarded and the filtrate was retained. 滤液 :V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层 :V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4 The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator.

[0146] (3) Dissolve 1 g of the product modified with double bonds in 10 mL of H 2 In a mixture of 1 mL of H O and 15 mL of anhydrous methanol, 1 g of GSH was dissolved in 3 mL of H 2 O and 2mL of anhydrous methanol; mix the above two solutions, add 50mg of azobisisobutyronitrile, and pass N 2 The reaction was carried out in a vacuum, and the mixture was stirred in an oil bath under magnetic stirring at 60° C. for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, and a solid was obtained by vacuum rotary evaporation. The obtained product was vacuum dried at 40° C. for 16 h to obtain adamantane modified with GSH (Ad-GSH), which was stored in a refrigerator.

[0147] (4) 2gFeCl 3 6H 2 O, 5.4 g NaAc was dissolved in 38 mL ethylene glycol, mixed and magnetically stirred for 1 h, transferred to a high temperature reactor, reacted at 200 ° C for 16 h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and vacuum dried at 50 ° C to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs).

[0148] (5) 45 mg of MNPs were added to 3.45 mL of 0.5 M NaCl aqueous solution, and after ultrasonic dispersion, 0.72 mL of anhydrous dimethyl sulfoxide was added, followed by 0.27 mL of 100 mM Ti(SO 4 ) 2 Then, 0.27 mL of 100 mM phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution. The mixture was transferred to a 23°C water bath, and 3 mL of 20 mM 3-(N-morpholinyl)propanesulfonic acid was added. The mixture was mechanically stirred for 3 h under the above conditions, and then heated with H 2 The resulting product was washed twice with O and ethanol respectively and dried in vacuum at 50 °C for 16 h to obtain the magnetic metal-polyphenol network (mTiCD).

[0149] (6) Disperse 30 mg Ad-GSH in 50 mL anhydrous dimethyl sulfoxide and dissolve 15 mg mTiCD in 10 mL H 2 O, and then the two solutions were mixed and ultrasonicated for 10 min. The mixture was then stirred in a 23 °C water bath for 3 h, washed once with dimethyl sulfoxide and twice with ethanol, and vacuum dried at 40 °C for 16 h to obtain the magnetic metal-polyphenol network material mTiCD@AG based on the host-guest interaction between β-CD and adamantane.

[0150] Example 5 The application of magnetic materials in separating and enriching proteins specifically includes:

[0151] (1) Separation and enrichment of glycosylated peptides

[0152] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0153] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0154] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2 The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0155] (2) Separation and enrichment of phosphorylated proteins

[0156] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0157] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0158] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0159] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0160] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2 O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0161] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0162] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0163] Example 6

[0164] A method for preparing a magnetic metal-polyphenol network material based on β-CD comprises the following steps:

[0165] (1) 2.28 g of β-CD and 1.62 g of N,N′-carbonyldiimidazole were dissolved in 10 mL of anhydrous N,N-dimethylformamide, and the two solutions were mixed after being fully dissolved, and reacted in a water bath at 23°C for 12 h. Then, 1.9 g of dopamine hydrochloride and 2.8 mL of triethylamine were added to the reaction solution in sequence, and the reaction was continued in a water bath at 23°C for 16 h. After the reaction was completed, the obtained solution was added dropwise to 400 mL of acetone, allowed to stand at room temperature for 1 h, and then centrifuged (10000 rpm, 5 min) to separate the white precipitate, and then the white precipitate was dispersed again with acetone, and then centrifuged under the same conditions. The obtained product was vacuum dried at 50°C for 16 h to obtain dopamine-modified β-CD (phen@CD).

[0166] (2) Dissolve 5.4 g of 1-carboxyadamantane in 150 mL of anhydrous dimethyl sulfoxide and add 5 g of K 2 CO 3 , stirred in a water bath with magnetic stirring at 23°C for 24 hours, after the reaction, the reaction product was filtered, the precipitate was discarded and the filtrate was retained. 滤液:V HCl =3:1 ratio to add 2mol / L dilute hydrochloric acid to the filtrate, and then use ethyl acetate and hexane mixture as the extractant to extract the filtrate in batches (V 乙酸乙酯 :V 己烷 =3:1, V 滤液 :V 萃取剂 =6:1), after standing for stratification, retain the upper organic layer liquid, and then add H 2 O(V 有机层 :V H2O =1:1), mix well and let stand for stratification, retain the upper organic layer liquid. Mix all the organic layer liquids after extraction, add excess Na 2 SO 4 The upper layer of liquid was dried and vacuum evaporated to obtain a light yellow liquid, i.e. adamantane modified with double bonds, which was stored in a refrigerator.

[0167] (3) Dissolve 1 g of the product modified with double bonds in 10 mL of H 2 In a mixture of 1 mL of H O and 15 mL of anhydrous methanol, 1 g of GSH was dissolved in 3 mL of H 2 O and 2mL of anhydrous methanol; mix the above two solutions, add 50mg of azobisisobutyronitrile, and pass N 2 The reaction was carried out in a vacuum, and the mixture was stirred in an oil bath under magnetic stirring at 60° C. for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature, and a solid was obtained by vacuum rotary evaporation. The obtained product was vacuum dried at 40° C. for 16 h to obtain adamantane modified with GSH (Ad-GSH), which was stored in a refrigerator.

[0168] (4) 1.35 g FeCl 3 6H 2 O, 3.6 g NaAc was dissolved in 25 mL ethylene glycol, mixed and magnetically stirred for 1 h, transferred to a high-temperature reactor, reacted at 200 ° C for 16 h, washed with distilled water and anhydrous ethanol for 3-4 times respectively, and vacuum dried at 50 ° C to obtain Fe 3 O 4 Magnetic nanoparticles (MNPs).

[0169] (5) 75 mg of MNPs were added to 5.75 mL of 0.5 M NaCl aqueous solution, and after ultrasonic dispersion, 1.2 mL of anhydrous dimethyl sulfoxide was added, followed by 0.48 mL of 100 mM Ti(SO 4 ) 2Then, 0.48 mL of 100 mM phen@CD anhydrous dimethyl sulfoxide solution was added thereto, and ultrasonic dispersion was continued during the process of adding the solution. The mixture was transferred to a 23°C water bath, and 7.5 mL of 20 mM 3-(N-morpholinyl)propanesulfonic acid was added. The mixture was mechanically stirred for 3 h under the above conditions, and then heated with H 2 The resulting product was washed twice with O and ethanol respectively and dried in vacuum at 50 °C for 16 h to obtain the magnetic metal-polyphenol network (mTiCD).

[0170] (6) Disperse 30 mg Ad-GSH in 50 mL anhydrous dimethyl sulfoxide and dissolve 15 mg mTiCD in 10 mL H 2 O, and then the two solutions were mixed and ultrasonicated for 10 min. The mixture was then stirred in a 23 °C water bath for 4 h, washed once with dimethyl sulfoxide and twice with ethanol, and dried in vacuum at 40 °C for 16 h to obtain the magnetic metal-polyphenol network material mTiCD@AG based on the host-guest interaction between β-CD and adamantane.

[0171] Example 6 The application of magnetic materials in separating and enriching proteins specifically includes:

[0172] (1) Separation and enrichment of glycosylated peptides

[0173] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 5% H 2 O + 5% FA), add 200 μL trypsin-digested immunoglobulin G solution (1 mg / mL), and incubate at room temperature for 1 h;

[0174] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0175] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (50% ACN + 49.9% H 2 The enriched glycosylated peptides were eluted with 4% 4% FA (0.1% FA) and incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0176] (2) Separation and enrichment of phosphorylated peptides

[0177] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (85% ACN + 10% H 2 O + 5% TFA), add 200 μL of trypsin-digested β-casein (1 mg / mL), and incubate at room temperature for 1 h;

[0178] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0179] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (1% NH 3 ·H 2 O) Eluted and enriched phosphorylated peptides were incubated at room temperature for 1 h. After separation in an external magnetic field, the supernatant was taken for mass spectrometry.

[0180] (3) Simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides

[0181] Step 1: Disperse the pretreated mTiCD@AG (1 mg) in 200 μL of Loading buffer (90% ACN + 7% H 2 O + 3% FA), add 100 μL of trypsin-digested immunoglobulin G solution (1 mg / mL) and 100 μL of trypsin-digested β-casein (1 mg / mL) mixed solution, and incubate at room temperature for 1 h;

[0182] Step 2: Separate the incubated mTiCD@AG by an external magnetic field, discard the supernatant, wash three times with 200 μL Loading buffer, and discard the supernatant after magnetic separation;

[0183] Step 3: Wash the washed mTiCD@AG with 20 μL of extraction buffer (3% NH 3 ·H 2 O) Elute the enriched glycosylated peptides and phosphorylated peptides, incubate at room temperature for 1 hour, separate in an external magnetic field, and take the supernatant for mass spectrometry.

[0184] The above descriptions are only several embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: The following steps are involved: Step 1: β -CD and N,N′-carbonyldiimidazole are dissolved in solvents respectively, mixed and reacted in a water bath, and then dopamine hydrochloride and triethylamine are added to continue the reaction to obtain dopamine-modified β - CD; Step 2: Dissolve 1-adamantane carboxylic acid in a solvent, add K2CO3, heat in a water bath, stir magnetically, and then drop allyl bromide, and continue the reaction under the above conditions to obtain adamantane Ad containing a double bond; Step 3: Dissolve reduced glutathione in a mixture of H2O and anhydrous CH3OH, then add it to the solution of adamantane containing double bonds obtained in step 2, add 2,2'-azobisisobutyronitrile and react in an oil bath vacuum to obtain GSH-modified Ad; Step 4: FeCl3•6H2O, NaAc and ethylene glycol are mixed and stirred evenly and then transferred to a reactor for reaction to obtain Fe3O4 magnetic nanoparticles; Step 5: Add the Fe3O4 magnetic nanoparticles obtained in step 4 to the NaCl aqueous solution, disperse them by ultrasonication, and then add anhydrous dimethyl sulfoxide, Ti(SO4)2 aqueous solution, and dopamine-modified Fe3O4 nanoparticles obtained in step 1 in sequence under ultrasonication. β -CD anhydrous DMSO solution, 3-(N-morpholinyl)propanesulfonic acid aqueous solution, heated in a water bath and stirred to react to obtain magnetic cyclodextrin-Ti 4+ Metal-polyphenol network materials; Step 6: Disperse the GSH-modified Ad obtained in step 3 in a solvent, and add the magnetic cyclodextrin-Ti prepared in step 5 4+ The metal-polyphenol network material aqueous solution is ultrasonically dispersed, and then transferred to a water bath, heated and stirred to react, to obtain a metal-polyphenol network material aqueous solution. β -Magnetic metal-polyphenol network material for CD.

2. A method according to claim 1 based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: In step 1 β The mass ratio of -CD, N,N′-carbonyldiimidazole and dopamine hydrochloride is 2.28:1.62:1.9 。 3. A method according to claim 1 based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: In the step 2, the mass ratio of 1-adamantanecarboxylic acid to K2CO3 is 5.4:

5.

4. A method according to claim 1 based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: In the step 3, the vacuum reaction temperature is 60-63°C and the reaction time is 22-24 h.

5. A method according to claim 1 based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: The reaction temperature of step 4 is 180-200°C and the reaction time is 14-16 h.

6. A method according to claim 1 based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: In the step 5, Fe3O4 magnetic nanoparticles mg: NaCl aqueous solution mL: anhydrous dimethyl sulfoxide mL: Ti(SO4)2 aqueous solution mL: dopamine modified β -CD anhydrous DMSO solution mL: 3-(N-morpholino)propanesulfonic acid aqueous solution mL is (30-90): (2.3-6.9): (0.48-1.44): (0.18-0.54): (0.18-0.54): (3-9).

7. A method according to claim 1 based on β -A method for preparing a magnetic metal-polyphenol network material of cyclodextrin, characterized in that: The reaction temperature of step six is ​​21-23°C, and the reaction time is 2-4 h.

8. The preparation method according to claim 1 obtained based on β -Magnetic metal-polyphenol network material for CD.

9. The method according to claim 8 β -CD magnetic metal-polyphenol network material, characterized in that, The basis of β -CD's magnetic metal-polyphenol network material is spherical in appearance, with a size concentrated in the range of 230-260 nm and paramagnetic.

10. The method according to claim 8 β -CD magnetic metal-polyphenol network material for separate and simultaneous separation and enrichment of glycosylated peptides and phosphorylated peptides.

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