Metal ion functionalized microsphere, preparation method and phosphorylated protein enrichment method

Abstract

The invention provides a phosphorylated protein enrichment method, and relates to the fields of materials, analytical chemistry and life science. The enrichment method of the phosphorylated protein comprises the following steps: dissolving a phosphorylated protein sample in a sample loading solution, mixing with super-hydrophilic metal ion functionalized microspheres, hatching, centrifuging, and discarding supernatant to obtain a super-hydrophilic material enriched with the phosphorylated protein; mixing the material enriched with the phosphorylated protein with an elution solution, incubating, and centrifuging to obtain enriched liquid of the phosphorylated protein; the super-hydrophilic metal ion functionalized microsphere is provided with a super-hydrophilic nano convex shell which is completely coated on the solid core. The enrichment method provided by the invention can overcome the non-specific adsorption problem caused by modification defects of a traditional monomolecular layer modified functional material, and has excellent enrichment selectivity; the method can overcome the limitation of an immunoprecipitation enrichment method antibody, has wide applicability, and is hopeful to occupy an important position in phosphorylated protein separation and enrichment.

Description

Technical Field

[0001] The present invention relates to the fields of material analysis chemistry and life sciences, and in particular to a method for enriching phosphorylated proteins based on superhydrophilic metal ion-functionalized microspheres Background Art

[0002] Protein phosphorylation is one of the most common and important post-translational modifications in organisms, so the research on phosphoproteomics has attracted extensive attention from many scientists around the world. However, due to the extremely low absolute content of phosphorylated proteins in organisms, the premise of phosphoproteomics research is the separation and enrichment of phosphorylated proteins. At present, due to the difficulty in separating intact phosphorylated proteins, phosphoproteomics research mainly relies on a "bottom-up" strategy, that is, first enzymatically digesting proteins into polypeptide segments without discrimination, identifying the phosphorylation sites at the peptide segment level, and then performing database searching, inference, and splicing to obtain complete phosphorylated protein information. Although this method is simple and feasible, there is a large gap between the phosphorylated information at the protein level obtained and the real information in actual biological samples. Therefore, directly separating at the protein level has always been a difficult problem that phosphoproteomics researchers have tried hard to solve but have not yet solved Summary of the Invention

[0003] The present invention provides an enrichment material with significant recognition ability for phosphorylated proteins for the separation and enrichment of phosphorylated proteins to solve the above technical problems. This material provides a method for separating and enriching phosphorylated proteins with high selectivity, high adsorption capacity, simple operation, and good repeatability, and can selectively enrich and separate phosphorylated proteins with relatively low content in biological samples

[0004] The technical solution of the present invention is as follows

[0005] The application method of the superhydrophilic metal ion-functionalized material in the enrichment and separation of phosphorylated proteins specifically adopts the solid-phase extraction mode (SPE) or the dispersive solid-phase extraction mode (dSPE) to enrich phosphorylated proteins, and the specific steps are as follows

[0006] When enriching phosphorylated proteins in the SPE mode, load the sample onto an SPE column filled with the superhydrophilic metal ion-functionalized material, and wash the material with a washing solution to remove non-phosphorylated proteins thereon; then elute the phosphorylated proteins with an elution solution; or when enriching and purifying phosphorylated proteins in the dSPE mode, directly mix the sample with the superhydrophilic metal ion-functionalized material, centrifuge, and discard the supernatant; wash the material with a washing solution, centrifuge, and remove non-phosphorylated proteins thereon; then wash with an elution solution, centrifuge, and collect the phosphorylated proteins

[0007] In the above solution, the metal ions of the superhydrophilic metal ion-functionalized microspheres include but are not limited to Cu 2+ , Zn2 + , Fe 3+ , Ga 3+ , Al 3+ , Ti 4+ , Zr 4+ One or more of them are mixed in any proportion;

[0008] In the above solution, the nano-protrusion shell outside the super-hydrophilic metal ion-functionalized microspheres is composed of a negatively charged hydrophilic polymer, and the inner core is composed of a hydrophobic polymer, having a solid and dense structure. The hydrophobic matrix is not exposed to the external environment, avoiding non-specific adsorption of external protein molecules to the hydrophobic matrix;

[0009] In the above solution, the sample loading solution and the eluent are a mixed solution of an additive, a buffer salt solution and an organic solvent. The volume percentage of the organic solvent is 0 - 30%, the concentration of the buffer salt solution is 0 - 200 mmol / L, the volume concentration of the additive is 0% - 10%, and the pH is in the range of 3 - 7; the eluent is a mixed solution of an additive, a buffer salt solution and an organic solvent. The volume percentage of the organic solvent is 0 - 30%, the concentration of the buffer salt solution is 0 - 200 mmol / L, the volume concentration of the additive is 0.01% - 10%, and the pH is in the range of 8 - 12;

[0010] Among them, the organic solvent is a mixture of one or more of acetonitrile, methanol, and ethanol; the buffer salt is a mixture of one or more of ammonium formate, ammonium acetate, ammonium bicarbonate, tris(hydroxymethyl)aminomethane (Tris), sodium hydrogen phosphate, and sodium dihydrogen phosphate; the additive in the sample loading solution is a mixture of one or more of formic acid, acetic acid, trifluoroacetic acid, and hydrochloric acid; the additive in the eluent is a mixture of one or more of ammonia water, sodium hydroxide, and potassium hydroxide.

[0011] The preparation method of the super-hydrophilic metal ion-functionalized microspheres. This preparation method uses the emulsion interfacial polymerization method to copolymerize hydrophilic monomers on the surface of the substrate composed of hydrophobic monomers. The specific preparation method is as follows:

[0012] 1) Disperse polystyrene microspheres into the emulsion obtained by ultrasonic emulsification of an organic solvent, a surfactant, and water, and stir;

[0013] 2) Mix hydrophilic monomers, a surfactant, hydrophobic monomers, an initiator, and water and perform ultrasonic emulsification;

[0014] 3) Add the emulsion obtained in step 2) to 1) and stir;

[0015] 4) Pass nitrogen into the emulsion obtained in step 3) to remove oxygen and polymerize;

[0016] 5) Wash the solid obtained in step 4) repeatedly with deionized water and ethanol, and then freeze-dry it.

[0017] 6) Disperse the solid obtained in step 5) in an aqueous solution of a metal compound, and stir overnight at room temperature. Wash the resulting microspheres three times with 0.1% aqueous TFA solution and store them in 0.1% aqueous TFA solution to obtain the product.

[0018] The hydrophilic monomer is one or more of sodium p-(4-vinylphenyl)phosphonate, sodium p-[(4-vinylphenyl)methyl]phosphonate, sodium vinylphosphonate, sodium allylphosphonate, sodium 1-methylvinylphosphonate, sodium 2-methyl-2-propenylphosphonate, and sodium (1-hydroxy-2-propenyl)phosphonate, and its concentration is 0.001 mol / L to 10 mol / L.

[0019] The hydrophobic monomer is one or more of styrene, α-methylstyrene, o-chlorostyrene, divinylbenzene, 4-vinylbiphenyl, methyl methacrylate, ethyl methacrylate, trichloroethylene, 1,2-dichloroethylene, hexafluorobutyl acrylate, hexafluorobutyl methacrylate, 9-vinylanthracene, 2-vinylnaphthalene, 9-vinylcarbazole, vinylcyclohexane, 4-aminostyrene, 4-bromostyrene, vinyl n-butyl ether, and cyclohexyl vinyl ether, and its concentration is 0.001 mol / L to 10 mol / L.

[0020] The diameter of the polystyrene microspheres of the polystyrene microspheres is 0.1 μm to 20 μm, and the mass concentration of the polystyrene microspheres in the resulting solution is 0.1 mg / mL to 1000 mg / mL; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, dodecyl ethoxysulfobetaine, cetyltrimethylammonium bromide, potassium monolauryl phosphate, higher alcohol phosphate disodium salt, span, tween, triton, polyvinyl alcohol, polyethylene glycol, and polyethylene glycol monomethyl ether, and its concentration is 0.00001 g / mL to 0.1 g / mL; the organic solvent is one or more of benzene, toluene, xylene, hexane, heptane, octane, decane, hexadecane, chlorobenzene, dichloromethane, chloroform, chlorododecane, bromododecane, and aniline, and its volume fraction is 0.001% to 1%; the initiator is one or more of azobisisobutyronitrile, azobis(2,4-dimethylvaleronitrile), azobiscyclohexanecarbonitrile, benzoyl peroxide, and tert-butyl peroxybenzoate, and its concentration is 0.001 mol / L to 0.2 mol / L.

[0021] The aqueous solution of the metal compound is one or more of CuCl2, Cu(NO3)2, CuSO4, ZnCl2, ZnSO4, FeCl3, Ga(NO3)3, AlCl3, Al2(SO4)3, Al(NO3)3, Ti(SO4)2, TiCl4, Zr(SO4)2, Zr(NO3)4, which are mixed in any proportion, and the concentration of the aqueous solution of the metal compound is 0.001 mol / L to 10 mol / L.

[0022] The volume ratio of the emulsion obtained in step 3) of step 2) to the solution obtained in step 1) is 0.5 to 50:1; the stirring in steps 1) and 4) is carried out at 10°C to 40°C for 0.5 h to 48 h. The emulsification time in steps 1-3) is 5 s to 1 h; the time for introducing nitrogen in step 5) is 5 min to 1 h, the polymerization temperature is 50°C to 100°C, and the time is 1 h to 48 h.

[0023] The enrichment method provided by the present invention can overcome the non-specific adsorption problem caused by the modification defects of traditional monolayer-modified functional materials, and has excellent enrichment selectivity; it can overcome the limitations of antibodies in the immunoprecipitation enrichment method, has wide applicability, and is expected to play an important role in the separation and enrichment of phosphorylated proteins.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The method for enriching phosphorylated proteins based on superhydrophilic metal ion-functionalized microspheres provided by the present invention exhibits characteristics such as high selectivity, high coverage, and high throughput when separating and enriching phosphorylated proteins, and can effectively separate and enrich phosphorylated proteins.

[0026] 2. The superhydrophilic metal ion-functionalized microspheres prepared by the present invention can be easily packed into columns with different lengths and inner diameters, and can also be directly added to centrifuge tubes, with simple operation and easy repetition. It is particularly suitable for the separation and enrichment of phosphorylated proteins in trace biological samples.

[0027] 3. The present invention has developed a series of metal ion-functionalized receptors based on hydrophilic phosphonates, and through the method of emulsion interfacial polymerization, a superhydrophilic nano-protrusion structure surface with a thickness ranging from ten to several hundred nanometers has been obtained. By organically combining the metal ion-functionalized superhydrophilic microspheres with the column solid-phase extraction mode or the dispersive solid-phase extraction mode, high-selectivity, high-coverage, and high-throughput enrichment of phosphorylated proteins in complex mixtures can be achieved, thereby realizing the selective enrichment of phosphorylated proteins in complex samples, and providing a new idea for the path of separating phosphorylated proteins. Therefore, it is expected to be widely used in aspects such as phosphorylated protein enrichment and large-scale separation. Description of the Drawings

[0028] Figure 1 Scanning electron microscope photograph of the superhydrophilic metal ion-functionalized microspheres prepared in Example 1 of the present invention.

[0029] Figure 2 Cross-sectional transmission electron microscope photograph of the superhydrophilic metal ion-functionalized microspheres prepared in Example 1 of the present invention.

[0030] Figure 3 X-ray photoelectron spectroscopy analysis chart of the superhydrophilic metal ion-functionalized microspheres prepared in Example 1 of the present invention.

[0031] Figure 4 Schematic diagram of the water contact angle of the superhydrophilic metal ion-functionalized microspheres prepared in Example 1 of the present invention.

[0032] Figure 5 Schematic diagram of the adsorption of phosphorylated protein by the superhydrophilic metal ion-functionalized microspheres prepared in Example 2 of the present invention.

[0033] Figure 6 One of the SDS-PAGE schematic diagrams of β-casein protein after enrichment by superhydrophilic metal ion-functionalized microspheres using the SPE and centrifugation modes.

[0034] Figure 7 Another SDS-PAGE schematic diagram of β-casein protein after enrichment by superhydrophilic metal ion-functionalized microspheres using the centrifugation mode. Detailed implementation manners

[0035] To make the content, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described below in conjunction with specific embodiments and drawings. These embodiments are only used to illustrate the present invention, and the present invention is not limited to the following embodiments.

[0036] Raw materials and equipment used in the examples:

[0037] Sodium dodecyl sulfate (SDS), 1-chlorodecane (CD), styrene (St), divinylbenzene (DVB), 2,2′-azobisisobutyronitrile (AIBN), and polyvinyl alcohol (PVA) were purchased from J&K scientific in Beijing, China. Sodium vinylbenzene phosphonate (SVBP) was ordered from Yintai (a Chinese chemical synthesis company in Dalian, China). β-casein and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. Titanium(IV) sulfate (Ti(SO4)2) was purchased from Sinopharm Chemical Reagent Co., Ltd. in China (≥99.9%, Shanghai, China). CY5-labeled β-casein (CY5-β-casein) and fluorescein isothiocyanate-labeled BSA (FITC-BSA) were self-made and purchased from Beijing Zhongke Chenyu Technology Co., Ltd. 10% SDS-PAGE microgels (10 wells, 10.0 cm × 10.0 cm). Tris-HCl buffer (pH = 6, 10 mM) was prepared by diluting 1 M Tris (tris(hydroxymethyl)aminomethane) buffer (pH = 6.5, from Yuanye Bio-Technology Co., Ltd. in Shanghai) with ultrapure water. Ultrapure water was from a milli Q system (Millipore, Bedford, MA, USA).

[0038] Application examples

[0039] Example 1

[0040] Preparation method of superhydrophilic metal ion-functionalized microspheres:

[0041] 1) Disperse 0.1 g of polystyrene microspheres (~2 μm) in 10 mL of SDS aqueous solution (0.25%, w / w), and add 5 mL of SDS (0.25%, w / w) aqueous solution containing 50 μL of CD, which has been ultrasonically emulsified for 30 s, to a 250 mL round-bottom flask, and stir at 40 °C for 20 h.

[0042] 2) Mix 0.6 g of hydrophilic monomer SVBP, 1.5 mL of hydrophobic monomer St, 0.025 mL of DVB, 20 mg of initiator AIBN, and 5 mL of aqueous solution containing 0.25% SDS (w / w), and ultrasonically emulsify for 30 s.

[0043] 3) Add the emulsion obtained in step 2) to 1), and stir at 40 °C for 20 h.

[0044] 4) Deoxygenate the mixture in step 3) by introducing nitrogen for 5 min. Immediately transfer the solution to an oil bath at 70 °C and keep it for 14 h. The obtained microspheres are washed 3 times with ethanol and deionized water and used after freeze-drying.

[0045] 5) Disperse the solid obtained in 4) in an aqueous solution of a metal compound and stir at room temperature overnight (12 h). Wash the obtained microspheres three times with 0.1% aqueous TFA solution and store them in 0.1% aqueous TFA solution.

[0046] Preferred metal compounds are FeCl3, Ti(SO4)2, and TiCl4. In this example, an aqueous solution of Ti(SO4)2 is selected, and its aqueous solution concentration is 500 mM; the mass of the solid contained in each 1 mL of the solution is 10 mg.

[0047] It can be seen that Figure 1-4 the hydrophobic monomers of the microspheres are dispersed in the oil phase of the emulsion, and the hydrophilic monomers are in the water phase. After heating, the hydrophilic and hydrophobic monomers copolymerize at the interface of the emulsion droplets. The hydrophobic monomers gradually crosslink inside the emulsion droplets to form a dense core; the hydrophilic monomers crosslink and polymerize inside and outside the emulsion droplets to form a shell with nanoscale protrusions. The diameter of the microspheres is 4 - 6 μm (average 5 μm), the surface has abundant nanoscale protrusions, and completely covers the substrate; the diameter of the nanoscale protrusions varies from 50 to 200 nm, and the average thickness of the nanoscale protrusion shell (including the protrusions) varies from 150 to 300 nm (average 250 nm). The signal of titanium ions can be seen in the XPS spectrum, indicating successful metal ion functionalization; the water contact angle schematic diagram shows the superhydrophilicity of the microspheres.

[0048] Example 2

[0049] Adjust the amount of the hydrophilic monomer SVBP during polymerization to 0.4 g, and keep other conditions the same as in Example 1. The material obtained after polymerization is characterized by SEM, TEM, XPS, and contact angle experiments. The experimental results show that microspheres with nanoscale protrusions on the surface can be obtained. The diameter of the microspheres is 4 - 6 μm (average 5 μm), the diameter of the nanoscale protrusions is slightly larger than that of the microspheres obtained in Example 1, ranging from 200 to 500 nm, and the average thickness of the nanoscale protrusion shell (including the protrusions) varies from 150 to 300 nm (average 250 nm). The signal of titanium ions can be seen in the XPS spectrum, indicating successful metal ion functionalization; the water contact angle schematic diagram shows the superhydrophilicity of the microspheres.

[0050] Example 3

[0051] When adjusting the amount of the hydrophilic monomer SVBP during polymerization to 0.5 g, with other conditions the same as in Example 1, the material obtained after polymerization was characterized by SEM, TEM, XPS and contact angle experiments. The experimental results showed that microspheres with nano-protrusions on the surface could be obtained. The diameter of the microspheres was 4 - 6 μm (average 5 μm), and the diameter of the nano-protrusions was slightly larger than that of the nano-protrusions of the microspheres obtained in Example 1, ranging from 200 to 300 nm. The average thickness of the nano-protrusion shell (including the protrusions) ranged from 150 to 300 nm (average 250 nm). The signal of titanium ions was visible in the XPS spectrum, indicating successful metal ion functionalization; the water contact angle schematic diagram demonstrated the superhydrophilicity of the microspheres.

[0052] Example 4

[0053] The superhydrophilic Ti 4+ functionalized microspheres were prepared according to the method described in Example 1. The adsorption behaviors of phosphorylated proteins and non-phosphorylated proteins on the superhydrophilic Ti 4+ functionalized microspheres were studied by laser scanning confocal microscopy (LSCM).

[0054] Using β-casein (from milk) as the model phosphorylated protein and BSA as the model non-phosphorylated protein.

[0055] The two proteins were modified with different fluorescent groups, and the microspheres adsorbed with proteins were characterized by laser scanning confocal microscopy. The specific process was as follows: Prepare a protein mixture of CY5-labeled β-casein (CY5-β-casein) and fluorescein isothiocyanate-labeled BSA (FITC-BSA), with the concentration of each protein being 0.2 mg / mL, and the solvent for dissolving the protein being an aqueous solution of 10 mM Tris / 10 mM NaCl with a pH value of 6. Subsequently, 0.3 mL of the protein mixture solution was mixed with 0.4 mg of the superhydrophilic Ti 4+ functionalized microspheres. After incubation for 3 min, the solution was characterized under a laser scanning confocal microscope.

[0056] It can be seen from Figure 5 that the superhydrophilic Ti 4+ functionalized microspheres strongly adsorbed β-casein and basically did not adsorb BSA, fully demonstrating the ability of this microsphere to specifically adsorb phosphorylated proteins.

[0057] Separation application example

[0058] Example 5

[0059] The superhydrophilic Ti 4+ functionalized microspheres were prepared according to the method described in Example 1.

[0060] Using the superhydrophilic Ti 4+Functionalized microspheres were used as enrichment materials to enrich phosphorylated proteins in the SPE mode. 1 mg of superhydrophilic Ti 4+ functionalized microspheres were loaded into a tip (a 1 - 200 μL pipette tip, here 150 μL). The enrichment material was rinsed with 50 μL of a solution containing 0.1% TFA by volume, and then equilibrated with 50 μL of a buffer of 10 mM Tris / 10 mM NaCl with a pH of 6. 1 μg of phosphorylated protein β-casein and 10 μg of interfering bovine serum albumin BSA were dissolved in 50 μL of a buffer of 10 mM Tris / 10 mM NaCl with a pH of 6 and loaded onto the enrichment material (hereinafter referred to as sample loading). After sample loading, the enrichment material was rinsed with 100 μL of a buffer of 10 mM Tris / 10 mM NaCl with a pH of 6 to remove non-phosphorylated proteins. Finally, 30 μL of 10% ammonia water (w / w) was used to elute the phosphorylated proteins from the material, and phosphorylated proteins were obtained with a recovery rate exceeding 80%. The identification of protein samples (including standard protein pure samples and different fractions separated from protein mixed samples by the enrichment material) was carried out by SDS-PAGE. The results showed that phosphorylated protein β-casein could be effectively enriched under the interference of 10-fold (mass multiple) non-phosphorylated protein BSA.

[0061] Example 6

[0062] The superhydrophilic Ti 4+ functionalized microspheres prepared by the method described in Example 1 were used as enrichment materials to enrich phosphorylated proteins in the dSPE mode. Activation and equilibration of the superhydrophilic Ti 4+ functionalized microspheres: 1.0 mg of the microspheres were loaded into a centrifuge tube, and 100 μL of an activation solution containing 0.1% TFA by volume was added. The enrichment material was completely dispersed in this solution by vortexing and shaking, left standing for 10 minutes and then centrifuged. The supernatant was discarded, and the precipitate was collected. Then 100 μL of an equilibration solution of 10 mM Tris / 10 mM NaCl with a pH of 6 was added, and the microspheres obtained by repeating the above process were used for the following experimental operations.

[0063] 1 μg of phosphorylated protein β-casein and 50 μg of interfering bovine serum albumin BSA were dissolved in 100 μL of a buffer of 10 mM Tris / 10 mM NaCl with a pH of 6. The protein sample was mixed with 1 mg of the activated and equilibrated enrichment material, incubated for 30 min, centrifuged, and the supernatant was discarded. The precipitate was incubated with 100 μL of a solution of 10 mM Tris / 10 mM NaCl (pH = 6) for 5 min, centrifuged, and the supernatant was removed. This step was repeated twice, and the two supernatants were combined. Then the precipitate was mixed with 50 μL of a solution of 10% ammonia water (w / w) and incubated for 5 min, centrifuged, and the supernatant was collected. This step was repeated once, and the two supernatants were combined.

[0064] The identification of various protein samples (including standard pure protein samples and different fractions of protein mixed samples after separation by enrichment materials) was carried out by SDS-PAGE. The results showed that more than 80% of the phosphorylated protein β-casein could be effectively enriched under the interference of 50-fold (mass fold) non-phosphorylated protein BSA.

[0065] Example 7

[0066] Superhydrophilic Ti 4+ functionalized microspheres were prepared by the method described in Example 1 and used as enrichment materials to enrich phosphorylated proteins in the dSPE mode. Superhydrophilic Ti 4+ Activation and equilibration of functionalized microspheres: 1.0 mg of microspheres were loaded into a centrifuge tube, 100 μL of activation solution containing 0.1% (v / v) TFA was added, and the enrichment materials were completely dispersed in the solution by vortexing and oscillation. After standing for 10 minutes, centrifugation was carried out, and the supernatant was discarded to collect the precipitate. Then 500 μL of equilibration solution of 10 mM Tris / 10 mM NaCl with a pH of 6 was added, and the microspheres obtained by repeating the above process were used for the following experimental operations.

[0067] 1 μg of phosphorylated protein β-casein and 100 μg of interfering bovine serum albumin BSA were dissolved in 100 μL of buffer of 10 mM Tris / 10 mM NaCl with a pH of 6. The protein sample was mixed with 1 mg of activated and equilibrated enrichment materials, incubated for 30 min, centrifuged, and the supernatant was discarded. The precipitate was incubated with 500 μL of solution of 10 mM Tris / 10 mM NaCl (pH = 6) for 5 min, centrifuged, and the supernatant was removed. This step was repeated twice, and the two supernatants were combined. Then the precipitate was mixed with 100 μL of solution of 10% ammonia water (w / w) and incubated for 5 min, centrifuged, and the supernatant was collected. This step was repeated once, and the two supernatants were combined.

[0068] The identification of various protein samples (including standard pure protein samples and different fractions of protein mixed samples after separation by enrichment materials) was carried out by SDS-PAGE. The results showed that more than 80% of the phosphorylated protein β-casein could be effectively enriched under the interference of 100-fold (mass fold) non-phosphorylated protein BSA.

[0069] From Figure 6-7 It can be seen that whether β-casein / BSA (w / w) is 1:10, 1:50 or 1:100, the protein samples obtained after enrichment and separation by superhydrophilic Ti 4+ functionalized microspheres can obtain β-casein protein by SDS-PAGE analysis and identification. It shows that the enrichment materials can selectively separate and purify phosphorylated proteins in complex systems.

[0070] In summary, the enrichment material of the present invention has excellent selective enrichment performance for phosphorylated proteins. Compared with conventional metal affinity materials modified with a single molecular layer, the superhydrophilic metal ion functional material has more excellent anti-nonspecific adsorption ability and higher selectivity when separating phosphorylated proteins. At the same time, compared with the traditional immunoprecipitation method, it has the advantages of low cost and wide application range, and has a higher coverage when enriching phosphorylated proteins. Therefore, it can be applied to the selective separation of large-scale and high-throughput phosphorylated proteins in complex systems. Combined with detection means such as liquid chromatography and liquid chromatography-mass spectrometry, it has broad application prospects in the field of phosphoproteomics research and other fields.

Claims

1. A metal ion-functionalized microsphere, characterized in that, The microspheres include a hydrophobic polymer core and a shell with nano-protrusions formed by the copolymerization of hydrophilic monomers and hydrophobic monomers on the surface of the hydrophobic polymer core; The hydrophilic monomers are one or more of sodium p-(4-vinylphenyl)phosphonate, sodium p-[(4-vinylphenyl)methyl]phosphonate, sodium vinylphosphonate, sodium allylphosphonate, sodium 1-methylvinylphosphonate, sodium 2-methyl-2-propenylphosphonate, and sodium (1-hydroxy-2-propenyl)phosphonate; The hydrophobic monomers are one or more of styrene, α-methylstyrene, o-chlorostyrene, divinylbenzene, 4-vinylbiphenyl, methyl methacrylate, ethyl methacrylate, trichloroethylene, 1,2-dichloroethylene, hexafluorobutyl acrylate, hexafluorobutyl methacrylate, 9-vinylanthracene, 2-vinylnaphthalene, 9-vinylcarbazole, vinylcyclohexane, 4-aminostyrene, 4-bromostyrene, vinyl n-butyl ether, and cyclohexyl vinyl ether; 2. The microsphere according to claim 1, characterized in that, The diameter of the superhydrophilic microspheres is 1 μm to 100 μm; the average diameter of the nano-protrusions is 10 nm to 1000 nm; the average thickness of the nano-protrusion shell (including the protrusions) is 10 nm to 5000 nm; The hydrophobic polymer is one or more of polystyrene, polydivinylbenzene, poly-9-vinylanthracene, poly-α-methylstyrene, poly-4-aminostyrene, poly-1,2-dichloroethylene, poly-o-chlorostyrene, poly-4-vinylbiphenyl, poly-methyl methacrylate, poly-ethyl methacrylate, poly-trichloroethylene, poly-hexafluorobutyl acrylate, poly-hexafluorobutyl methacrylate, poly-vinyl n-butyl ether, poly-2-vinylnaphthalene, poly-9-vinylcarbazole, poly-vinylcyclohexane, poly-4-bromostyrene, and poly-cyclohexyl vinyl ether or a copolymer of two or more of the monomers constituting them; 3. The microsphere according to claim 1 or 2, characterized in that, The metal ions of the metal ion-functionalized microspheres include, but are not limited to, Cu 2+ , Zn 2+ , Fe 3+ , Ga 3+ , Al 3+ , Ti 4+ , Zr 4+ One or more of them are mixed in any proportion.

4. The microsphere according to claim 1 or 2, characterized in that, The core has a solid and dense structure, and the outer shell is composed of a negatively charged hydrophilic polymer. The hydrophobic matrix is not exposed to the external environment, avoiding non-specific adsorption of external protein molecules to the hydrophobic matrix; the metal ion functionalized material is a superhydrophilic metal ion functionalized material with abundant superhydrophilic nano-protrusions and completely coated on the core, and the metal ions are fixed on the nano-protrusions; wherein, the hydrophilic polymer is a copolymer of one or more of poly-sodium p-(4-vinylphenyl)phosphonate, poly-sodium p-[(4-vinylphenyl)methyl]phosphonate, poly-vinylphosphonate, poly-allylphosphonate, poly-1-methylvinylphosphonate, poly-2-methyl-2-propenylphosphonate, and poly-(1-hydroxy-2-propenyl)phosphonate.

5. A method for preparing the metal ion-functionalized microsphere according to any one of claims 1-4, characterized in that, The superhydrophilic metal ion functionalized microspheres are synthesized by the emulsion interfacial polymerization method, including the following steps: 1) Dispersing the hydrophobic polymer microspheres serving as the core into an emulsion obtained by ultrasonic emulsification of an organic solvent, a surfactant, and water, and stirring; 2) Mixing and ultrasonic emulsifying the hydrophilic monomers, hydrophobic monomers, initiator, surfactant, and water; 3) Adding the emulsion obtained in step 2) to 1) and stirring; 4) Passing nitrogen into the emulsion obtained in step 3) to remove oxygen and polymerizing; 5) Washing the solid obtained in step 4) with water and ethanol in sequence, and freeze-drying; 6) Disperse the solid obtained in step 5) in an aqueous solution of a metal compound, stir at room temperature for 8 - 24 hours, and perform solid-liquid separation to obtain microspheres.

6. The preparation method according to claim 5, characterized in that, The diameter of the hydrophobic polymer microspheres in step 1) is 0.1 μm - 20 μm, and the mass concentration of the hydrophobic polymer microspheres in the obtained solution is 0.1 mg / mL - 1000 mg / mL. Preferably, the diameter of the polymer microspheres is 0.1 μm to 10 μm, and the concentration is 1 mg / mL to 100 mg / mL. More preferably, the diameter of the polymer microspheres is 0.5 μm to 5 μm, and the concentration is 5 mg / mL to 20 mg / mL. The hydrophilic monomer in step 2) is one or more of sodium p-(4-vinylphenyl)phosphonate, sodium p-[(4-vinylphenyl)methyl]phosphonate, sodium vinylphosphonate, sodium allylphosphonate, sodium 1-methylvinylphosphonate, sodium 2-methyl-propenylphosphonate, and sodium (1-hydroxy-2-propenyl)phosphonate. Its concentration is 0.001 mol / L to 10 mol / L. Preferably, the concentration of the hydrophilic monomer is 0.05 mol / L to 2 mol / L. More preferably, the concentration of the hydrophilic monomer is 0.1 mol / L to 1 mol / L. The hydrophobic monomer in step 2) is one or more of styrene, α-methylstyrene, o-chlorostyrene, divinylbenzene, 4-vinylbiphenyl, methyl methacrylate, ethyl methacrylate, trichloroethylene, 1,2-dichloroethylene, hexafluorobutyl acrylate, hexafluorobutyl methacrylate, 9-vinylanthracene, 2-vinylnaphthalene, 9-vinylcarbazole, vinylcyclohexane, 4-aminostyrene, 4-bromostyrene, vinyl n-butyl ether, and cyclohexyl vinyl ether. Its concentration is 0.001 mol / L to 10 mol / L. Preferably, the concentration of the hydrophobic monomer is 0.1 mol / L to 5 mol / L. More preferably, the concentration of the hydrophobic monomer is 1 mol / L to 5 mol / L.

7. The preparation method according to claim 5, characterized in that, The surfactant is one or more of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, dodecyl ethoxysulfobetaine, cetyltrimethylammonium bromide, potassium monolauryl phosphate, disodium higher alcohol phosphate (C 16 H 13 OPO3Na2), Span, Tween, Triton, polyvinyl alcohol, polyethylene glycol, methoxypolyethylene glycol, and its concentrations in steps 1) and 2) are respectively 0.00001 g / mL to 0.1 g / mL, preferably 0.001 g / mL to 0.1 g / mL, and more preferably 0.005 g / mL to 0.05 g / mL; The organic solvent in step 1) is one or more of benzene, toluene, xylene, hexane, heptane, octane, decane, 1-chlorodecane, hexadecane, chlorobenzene, dichloromethane, chloroform, chlorododecane, bromododecane, and aniline. Its volume fraction is 0.001% to 1%. Preferably, the volume fraction is 0.01% to 1%. More preferably, the volume fraction is 0.05% to 0.5%. The initiator is one or more of azobisisobutyronitrile, azobisisoheptonitrile, azobicyclohexylcarbonitrile, benzoyl peroxide, and tert-butyl peroxybenzoate. Its concentration in step 2) is 0.001 mol / L to 0.2 mol / L. Preferably, the concentration is 0.005 mol / L to 0.2 mol / L. More preferably, the concentration is 0.01 mol / L to 0.1 mol / L. The aqueous solution of the metal compound is one or more of CuCl2, Cu(NO3)2, CuSO4, ZnCl2, ZnSO4, FeCl3, Ga(NO3)3, AlCl3, Al2(SO4)3, Al(NO3)3, Ti(SO4)2, TiCl4, Zr(SO4)2, Zr(NO3)4, which are mixed in any proportion. The concentration of the aqueous solution of the metal compound is 0.001 mol / L to 10 mol / L, preferably 0.01 mol / L to 1 mol / L, and more preferably 0.1 mol / L to 1 mol / L.

8. The preparation method according to claim 5, 6 or 7, characterized in that, The volume ratio of the emulsion obtained in step 2) to the solution obtained in step 1) is 0.5 to 50:1; The stirring in steps 1) and 3) is carried out at 10°C to 40°C for 0.5 h to 48 h respectively; The time of ultrasonic emulsification in steps 1) and 2) is 5 s to 1 h; The time of introducing nitrogen in step 4) is 5 min to 1 h; The polymerization temperature is 50°C to 100°C, preferably 60°C to 80°C, more preferably 65°C to 75°C, and the time is 1 h to 48 h, preferably 5 to 24 h, more preferably 12 to 16 h; The microspheres obtained in step 6) are washed 1-5 times with an aqueous solution of TFA with a volume concentration of 0.001% to 1%, and stored in an aqueous solution of TFA with a volume concentration of 0.001% to 1% to obtain the product.

9. A method for enriching phosphorylated proteins, characterized in that, Using the metal ion-functionalized microspheres described in any one of claims 1-4 as the stationary phase packing; 1) includes the following steps: Dissolve the phosphorylated protein sample in the loading solution and mix it with the superhydrophilic metal ion-functionalized microspheres, incubate, and discard the supernatant after centrifugation to obtain a superhydrophilic material enriched with phosphorylated protein; Mix the material enriched with phosphorylated protein with the elution solution, incubate, and centrifuge to discard the supernatant; Mix the material enriched with phosphorylated protein with the elution solution, incubate, and centrifuge to obtain an enriched solution of phosphorylated protein; Or 2), in the solid-phase extraction mode, pack the superhydrophilic metal ion-functionalized microspheres into an SPE small column with a frit at the end and rinse and equilibrate with the elution solution and the loading solution in turn; dissolve the protein sample in the loading solution and load it onto an SPE small column with a frit at the end filled with heavy superhydrophilic microspheres, rinse the microspheres adsorbed with phosphorylated protein with the loading solution, and then elute the phosphorylated protein adsorbed on the microspheres with the elution solution to prepare the product.

10. The method according to claim 9, wherein, The loading solution and the elution solution are composed of a mixture of an additive, a buffer salt solution and an organic solvent. The volume percentage of the organic solvent is 0-30%, the concentration of the buffer salt solution is 0-200 mmol / L, the volume concentration of the additive is 0%-10%, and the pH is in the range of 3-7; The elution solution is composed of a mixture of an additive, a buffer salt solution and an organic solvent. The volume percentage of the organic solvent is 0-30%, the concentration of the buffer salt solution is 0-200 mmol / L, the volume concentration of the additive is 0.01%-10%, and the pH is in the range of 8-12; Among them, the organic solvent is one or more mixtures of acetonitrile, methanol, and ethanol; The buffer salt is a mixture of one or more of ammonium formate, ammonium acetate, ammonium bicarbonate, tris(hydroxymethyl)aminomethane (Tris), disodium hydrogen phosphate, and sodium dihydrogen phosphate; The additive in the sample loading solution is a mixture of one or more of formic acid, acetic acid, trifluoroacetic acid, and hydrochloric acid; The additive in the eluent is a mixture of one or more of ammonia water, sodium hydroxide, and potassium hydroxide.

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