Preparation method of Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification

Abstract

The invention provides a preparation method of a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification, and belongs to the technical field of magnetic material synthesis. The method comprises the following steps: firstly, preparing MCeO2; the preparation method comprises the following steps: mixing MCeO2, hexadecyl trimethyl ammonium bromide and ammonia water to obtain a mixed solution, adding 1, 2-bis (triethoxysilane) ethane into the mixed solution for reaction to obtain a product, and dispersing the product into an NH4NO3 solution for reflux reaction to obtain Janus MCP; the preparation method comprises the following steps: reacting Janus MCP with a silane coupling agent, dissolving aminated Janus MCP and 4-ethylene sulfonyl benzoic acid in a buffer solution containing EDC and NHS, and reacting to obtain the Janus material. The preparation method is reliable, the material performance is stable, and the enrichment efficiency of the magnetic Janus on target peptide fragments (phosphorylated peptide fragments and palmitoylated peptide fragments) is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic material synthesis, and in particular relates to a method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications. Background Art

[0002] Janus nanomaterials have anisotropy in physical and chemical properties, and have broadened their application areas through various functional modifications, and have attracted much attention.

[0003] Phosphorylation modification cannot be ignored in proteomics research, and abnormal phosphorylated peptides are often used to distinguish and identify disease-related biomarkers. Similarly, dynamic and reversible palmitoylation modification is also one of the most important post-translational modifications, involving the covalent attachment of long-chain fatty acids (mainly 16-carbon palmitic acid) to specific cysteine ​​residues through thioester bonds, and plays an extremely important role in a wide range of biological processes such as cell signal transduction, apoptosis, and carcinogenesis.

[0004] At present, the commonly used enrichment methods for phosphorylation modification include anion exchange chromatography, immobilized metal ion affinity chromatography, metal oxide / hydroxide affinity chromatography and molecular imprinting. The ideal enrichment material should have good properties such as specific binding to phosphorylated peptides, easy modification, and convenient elution and separation. The research methods include "palmitate-centered", "cysteine-centered", bioinformatics prediction and mass spectrometry direct detection. However, due to the low abundance and stoichiometry of post-modified peptides in organisms, the lack of clear consensus sequences, the lack of commercial antibodies, and the serious signal suppression of a large number of non-modified peptides in mass spectrometry analysis, the idea of ​​fully characterizing protein phosphorylation and palmitoylation modifications still faces great analytical challenges, especially the lack of methods to achieve simultaneous screening of two important post-modified peptides. In view of this, before mass spectrometry analysis, the development of precise and efficient selective enrichment methods based on chemical strategies has become a prerequisite for exploring phosphorylation and palmitoylation proteomics.

[0005] Currently, there is no Janus material developed for the simultaneous screening of protein phosphorylation and palmitoylation modifications. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification provided by the present invention is reliable, the material performance is stable, and the enrichment efficiency of magnetic Janus for target peptides (phosphorylated peptides and palmitoylated peptides) is significantly improved.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications comprises the following steps: Step 1: Prepare CeO2-coated ferroferric oxide magnetic nanoparticles MCeO2; Step 2: Mix the MCeO2 obtained in step 1, hexadecyltrimethylammonium bromide and ammonia water to obtain a mixed solution, add 1,2-bis(triethoxysilane)ethane to the mixed solution for reaction to obtain a product, disperse the product in NH4NO3 solution for reflux reaction to obtain a mesoporous silicon in situ grown on magnetic CeO2. Janus material, referred to as Janus MCP; Step 3: The Janus MCP obtained in step 2 is reacted with a silane coupling agent to obtain an amino-modified Janus MCP. The amino-modified Janus MCP and 4-ethylenesulfonylbenzoic acid are dissolved in a buffer solution containing EDC and NHS to react to obtain a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications.

[0008] Preferably, the size of the ferroferric oxide magnetic nanospheres MCeO2 coated with CeO2 in step 1 is between 200 and 250 nm.

[0009] Preferably, in step 2, the mass mg of MCeO2: the mass mg of hexadecyltrimethylammonium bromide: the volume mL of ammonia water: the mass mg of 1,2-bis(triethoxysilane)ethane is 90:150:1.8:100.

[0010] Preferably, the reaction temperature for adding 1,2-bis(triethoxysilane)ethane for reaction in step 2 is room temperature, the rotation speed is 800 rpm, and the reaction time is 3 h.

[0011] Preferably, the reaction temperature of the reflux reaction in step 2 is 80-100° C., and the reaction time is 8-14 h.

[0012] Preferably, the concentration of the NH4NO3 solution in step 2 is 5.0-7.0 g / L.

[0013] Preferably, the silane coupling agent in step three is 3-aminopropyltriethoxysilane (APTES).

[0014] Preferably, the reaction temperature of the neutralization reaction of the silane coupling agent in step 3 is 50-65° C., and the reaction time is 20-24 h.

[0015] Preferably, in step 3, the mass ratio of the aminated Janus MCP, 4-ethylenesulfonylbenzoic acid, carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 13.5:3:60:80.

[0016] Preferably, the reaction temperature in step 3 is room temperature, and the reaction time is 8 to 14 hours.

[0017] Beneficial effects of the present invention The present invention provides a method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification. The preparation of the Janus material is to first synthesize CeO2-coated ferroferric oxide magnetic nano-microspheres, then use a template to control the nucleation growth of periodic mesoporous silica (PMO) on the MCeO2 material to form a Janus MCP, and finally use a chemical cross-linking method to modify 4-ethylenesulfonylbenzoic acid to the PMO unit of the Janus MCP to prepare the final Janus material. The Janus material of the present invention consists of a magnetic unit and a mesoporous silicon unit. A layer of metal oxide CeO2 with a bidentate structure that can form a bridge with a phosphate group under acidic conditions is coated on the surface of the magnetic unit as a matrix for screening phosphorylated peptides. In the mesoporous silicon unit, the unique structure of multiple modification sites of mesoporous silicon is used to modify the vinylsulfonyl group with affinity for palmitoylated peptides. The preparation method of the present invention is reliable, the material performance is stable, and the enrichment efficiency of the magnetic Janus for target peptides (phosphorylated peptides and palmitoylated peptides) is significantly improved. The magnetic adsorbent synthesized by the present invention is used as a matrix for magnetic solid phase microextraction (MSPE) technology, which is suitable for the analysis of phosphorylated peptides and palmitoylated peptides in complex biological samples (such as cell lysates), proving that it has excellent application potential in the separation of proteomics and the field of MSPE. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the infrared spectrum of MCeO2 and Janus MCP materials prepared in Example 1 of the present invention.

[0019] Figure 2 This is the Zeta potential diagram of MCeO2 and Janus MCP materials prepared in Example 1 of the present invention.

[0020] Figure 3 The scanning electron microscope images of the Janus MCP-BVA materials prepared in Example 1 of the present invention and Comparative Examples 1-2 are shown.

[0021] Figure 4 This is a scanning electron microscope image of MCeO2 prepared in Comparative Examples 3 and 4 of the present invention.

[0022] Figure 5Mass spectra of phosphorylated peptides and palmitoylated peptides detected for Janus MCP and Janus MCP-BVA materials prepared in Example 1 of the present invention, respectively. DETAILED DESCRIPTION

[0023] A method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications comprises the following steps: Step 1: Prepare CeO2-coated ferroferric oxide magnetic nanoparticles MCeO2; Step 2: Mix the MCeO2 obtained in step 1, hexadecyltrimethylammonium bromide and ammonia water to obtain a mixed solution, add 1,2-bis(triethoxysilane)ethane to the mixed solution for reaction to obtain a product, disperse the product in NH4NO3 solution for reflux reaction to obtain a mesoporous silicon in situ grown on magnetic CeO2. Janus material, referred to as Janus MCP; Step 3: The Janus MCP obtained in step 2 is reacted with a silane coupling agent to obtain an amino-modified Janus MCP. The amino-modified Janus MCP and 4-ethylenesulfonylbenzoic acid are dissolved in a buffer solution containing EDC and NHS to react to obtain a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications.

[0024] According to the present invention, the preparation method of the CeO2-coated ferroferric oxide magnetic nanospheres MCeO2 preferably comprises: Step (1-1), weighing FeCl3·6H2O and sodium acetate and dissolving them in a solvent, the solvent is preferably ethylene glycol, and uniformly dispersing them to obtain a reaction solution, then adding the reaction solution to a reactor for reaction, the reaction temperature is preferably 180°C, the reaction time is preferably 14h, to obtain a product, the obtained product is preferably washed alternately with ultrapure water and anhydrous ethanol, and dried to obtain Fe3O4 nanoparticles, the molar ratio of FeCl3·6H2O to sodium acetate is preferably 24:79.5; Step (1-2), weighing the above-mentioned Fe3O4 nanoparticles and dissolving them in a mixed solution containing ethanol, water and ammonia water, and uniformly dispersing them by ultrasonication, wherein the ultrasonication time is preferably 30 minutes, and slowly adding tetraethyl silicate (TEOS) to the above-mentioned solution for reaction, wherein the reaction temperature is preferably room temperature, and the reaction time is preferably 10 hours, to obtain Fe3O4@SiO2; the mass mg of the Fe3O4 nanoparticles: the volume mL of ethanol: the volume mL of water: the volume mL of ammonia water: the volume mL of tetraethyl silicate are preferably 175:110:28:0.7:0.5; Step (1-3), dispersing the above Fe3O4@SiO2 in ultrapure water, ultrasonic treatment, the ultrasonic treatment time is preferably 30 min, then adding Ce(NO3)3·6H2O and urea to the above mixture, stirring and mixing, adding to the reactor for reaction, the reaction temperature is preferably 120°C, the reaction time is preferably 8h, and then separated by magnetic absorption, and then repeatedly washing the brown-yellow solid with ethanol and water, and vacuum drying to obtain a CeO2-coated magnetic material, named MCeO2. The mass ratio of the Fe3O4@SiO2, Ce(NO3)3·6H2O and urea is preferably 3:6:9. The size of the MCeO2 is between 200 and 250 nm; the present invention obtains a MCeO2 material of suitable size by adjusting the reaction temperature and reaction time in this step, so as to prepare for the subsequent synthesis of Janus material. When the reaction temperature is too high, the size of the synthesized MCeO2 is too large and is not suitable for subsequent growth into Janus. Similarly, when the reaction time is less than 8 h, the synthesized MCeO2 is too small and is not suitable for subsequent growth into Janus.

[0025] According to the present invention, MCeO2, hexadecyltrimethylammonium bromide and ammonia water obtained in step 1 are added to ultrapure water respectively, mixed and stirred at room temperature to make them dispersed evenly, the mixing time is preferably 30 min, and a mixed solution is obtained. 1,2-bis(triethoxysilane)ethane (BTEE) is added to the above mixed solution for reaction, the reaction temperature is preferably room temperature, the rotation speed is preferably 800 rpm, the reaction time is preferably 3 h, and after the reaction stops, it is washed and dried, the solvent is discarded, and the product is retained; the present invention obtains a Janus material of appropriate size by adjusting the rotation speed and reaction time in this step. When the rotation speed is too low, the size of the mesoporous silicon unit in the synthesized Janus material is too small, and a Janus with relatively consistent symmetry cannot be formed. When the reaction time is too long, the size of the mesoporous silicon unit in the synthesized Janus material is too long, and a Janus with good symmetry cannot be formed. The adsorption sites provided on the surface of the material prepared according to the optimal ratio can be effectively used for the enrichment of phosphorylated peptides and palmitoylated peptides in biological samples.

[0026] According to the present invention, the above product is dispersed in NH4NO3 solution, refluxed, and post-treated to obtain Janus MCP; the reaction temperature is preferably 80-100°C, more preferably 90°C, the reaction time is preferably 8-14h, more preferably 12h, the NH4NO3 solution is preferably an ethanol solution of NH4NO3, and the concentration of the NH4NO3 solution is preferably 5.0-7.0 g / L, more preferably 6.0 g / L. The post-treatment is preferably: after the above reflux liquid is cooled, centrifuge (11000 rpm) to remove the supernatant, alternately wash the obtained orange-yellow solid with ethanol and ultrapure water, and vacuum dry the obtained product at 60 °C for standby use. The mass mg of MCeO2: the mass mg of hexadecyltrimethylammonium bromide: the volume mL of ammonia water: the mass mg of 1,2-bis(triethoxysilane)ethane: the volume mL of NH4NO3 solution are preferably 90:150:1.8:100:150.

[0027] According to the present invention, the Janus MCP obtained above is preferably first dispersed in a solvent, and the solvent is preferably ethanol, to obtain a uniform solution, and the concentration of the solution is preferably 2 mg / mL, and then a silane coupling agent is added to the above solution for reaction, and the reaction temperature is preferably 50-65°C, and the reaction time is preferably 20-24h. After stopping the reaction, after magnetic separation and washing operations, the obtained product is dried to obtain an amino-modified Janus MCP; the silane coupling agent is preferably 3-aminopropyltriethoxysilane (APTES).

[0028] According to the present invention, EDC (carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are dissolved in a phosphate buffer solution, and the pH is adjusted to 6 to obtain a buffer solution containing EDC and NHS, and then the aminated Janus MCP and 4-ethylenesulfonylbenzoic acid (BVA) are dissolved in the buffer solution containing EDC and NHS to react, the reaction temperature is preferably room temperature, the reaction time is preferably 8 to 14 hours, more preferably 12 hours, and after magnetic separation, washing and drying, a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification is obtained, named Janus MCP-BVA. The mass ratio of the aminated Janus MCP, 4-ethylenesulfonylbenzoic acid, EDC and NHS is preferably 13.5:3:60:80.

[0029] The present invention uses Fe3O4 magnetic nanoparticles as one side of the Janus structure and as a carrier for magnetic solid phase extraction to pre-treat samples in proteomics, which facilitates elution and improves enrichment capacity; the other side is periodic mesoporous silica (PMO), and the porous structure provides abundant binding sites. BVA is modified onto PMO through NHS-EDC coupling reaction, and CeO2 is coated onto magnetic spheres by in-situ precipitation. Combined with MALDI-TOF-MS mass spectrometry analysis, dual enrichment of phosphorylated peptides and palmitoylated peptides is achieved.

[0030] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The raw materials involved in the embodiments are all commercially available. Example 1

[0031] A method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications comprises the following steps: Step (1), synthesis of CeO2-coated ferroferric oxide magnetic nanoparticles (named MCeO2): 3.24 g FeCl3·6H2O (24 mmol) and 6.52 g sodium acetate (79.5 mmol) were weighed and dissolved in 120 mL ethylene glycol and dispersed evenly. 60 mL of the above solution was transferred to a 100 mL polytetrafluoroethylene (PTFE) reactor and reacted in an oven at 180°C for 14 h. After cooling to room temperature, the obtained ferroferric oxide magnetic nanoparticles (Fe3O4) were separated by magnetic adsorption, washed alternately with ultrapure water and anhydrous ethanol for 5 times, and dried in a vacuum oven at 60 °C for use. 175 mg of Fe3O4 nanoparticles were weighed and dissolved in a mixed solution containing 110 mL ethanol, 28 mL water and 0.7 mL ammonia water, and ultrasonicated for 30 min to make it evenly dispersed. 0.5 mL tetraethyl silicate (TEOS) was slowly added and stirred at room temperature for 10 h. After the reaction stopped, it was washed and dried by the above method to obtain Fe3O4@SiO2. 300 mg Fe3O4@SiO2 was dispersed in 120 mL ultrapure water and treated with ultrasound for 30 min. Then, 0.6 g Ce(NO3)3·6H2O and 0.9 g urea were added to the mixture. After stirring and mixing evenly, it was transferred to a 100 mL reactor. After reacting at 120°C for 8 h, it was separated by magnetic adsorption. The brown-yellow solid was repeatedly washed with ethanol and water. After vacuum drying, the CeO2-coated magnetic material was obtained, named MCeO2.

[0032] Step (2), using a template to control the nucleation and growth of periodic mesoporous silica (PMO) in MCeO2 material under alkaline conditions to form a Janus material (named Janus MCP): Take 90 mg MCeO2, 150 mg hexadecyltrimethylammonium bromide (CTAB) and 1.8 mL ammonia water and add them to 100 mL ultrapure water respectively, stir at room temperature for 30 min to make it dispersed evenly. Add 1,2-bis(triethoxysilane)ethane (BTEE) dropwise to the mixture, adjust the speed to 800 rpm, react at room temperature for 3 h, after the reaction stops, wash and dry, discard the solvent, and retain the product. Disperse the product in 150 mL of ethanol solution containing 6.0 g / L NH4NO3, reflux at 90 °C overnight, after the reflux liquid is cooled, centrifuge (11000 rpm) to remove the supernatant, wash the orange-yellow solid with ethanol and ultrapure water alternately, and dry the obtained product in vacuum at 60 °C for use, named Janus MCP.

[0033] Step (3), using chemical cross-linking method, 4-vinylsulfonylbenzoic acid (BVA) was modified onto the PMO unit of Janus MCP (named as Janus MCP-BVA): The Janus MCP material was dispersed in ethanol to form a uniform solution of 2 mg / mL. 50 mL of the solution was added with 0.2 mL of silane coupling agent (ATPES) and reacted at 50 °C for 24 h. After stopping the reaction, the product was dried in a vacuum oven at 60 °C after magnetic separation and washing to obtain the amino-modified Janus MCP. 60 mg of EDC and 80 mg of NHS were dissolved in 20 mL of phosphate buffer solution and the pH was adjusted to 6. 13.5 mg of amino-modified Janus MCP and 3 mg of BVA were added to the above buffer solution and stirred overnight at room temperature in the dark. After stopping the reaction, the product was dried in a vacuum oven at 60 °C after magnetic separation and washing to obtain the final product, named Janus MCP-BVA.

[0034] Figure 1 The infrared spectra of MCeO2 and Janus MCP materials prepared in Example 1 of the present invention, wherein curve (a) represents MCeO2, and curve (b) represents Janus MCP material. In curve (a), at ~580 cm -1 The peaks at 1075 and 845 cm are attributed to the lattice vibration of Fe3O4; -1 The characteristic peaks at 1506 and 1411 cm -1The two peaks at 3450 cm-1 are related to the bending vibration of the hydroxyl groups on cerium oxide (Ce-OH); -1 The peak at ~580 cm corresponds to the stretching vibration of OH in water, indicating the smooth synthesis of MCeO2. -1 The peaks at 1506 and 1411 cm -1 The two Ce-OH peaks at 1041 and 916 cm -1 The two newly added peaks at , representing the Si-C peak and the Si-O-Si peak of linear polysiloxane, prove the successful synthesis of JanusMCP.

[0035] Figure 2 The zeta potential diagram of MCeO2 and Janus MCP materials prepared in Example 1 of the present invention is shown in FIG. Figure 2 It shows that because CeO2 is coated on the surface of the magnetic sphere under alkaline conditions, the hydroxyl groups it carries cause the potential to be negative; in the process of growing mesoporous silicon, the positively charged template agent hexadecylammonium bromide is adsorbed on the surface of the particles through electrostatic action, causing the potential to become positive. The results are consistent with the predictions, proving the successful synthesis of MCeO2 and Janus MCP materials.

[0036] Comparative Example 1 The experimental steps and conditions were the same as those in Example 1, except that the rotation speed in step (2) was adjusted to 200 rpm, and the other conditions remained unchanged.

[0037] Comparative Example 2 The experimental steps and conditions are the same as those in Example 1, except that the reaction time in step (2) is adjusted to 6 h at room temperature, and the other conditions remain unchanged.

[0038] Figure 3 The scanning electron microscope images of the Janus MCP-BVA materials prepared in Example 1 and Comparative Examples 1-2 of the present invention are shown in FIG. Figure 3 (a) represents comparative example 2, Figure 3 (b) represents comparative example 1, Figure 3 (c) represents Example 1. It can be seen from the figure that the mesoporous silicon portion of the Janus synthesized in Comparative Example 2 grew too long due to the long reaction time, and the size was larger than 1 μm, which was not suitable for the separation and analysis of the target analyte; while the mesoporous silicon portion of the Janus synthesized in Comparative Example 1 grew too small due to the slow rotation speed during the synthesis of the Janus. Figure 3(b) It can be seen that the formed Janus structure is obviously asymmetric. And due to the slow reaction speed, the magnetic material is seriously agglomerated, and the formed Janus is not suitable for separation and analysis of target analytes; while the reaction speed and reaction time of the Janus synthesized in Example 1 of the present invention are well optimized during the synthesis process. Figure 3 (c) It can be seen that the Janus morphology formed is uniform and has good symmetry. The Janus magnetic adsorbent obtained under the reaction conditions is very suitable for the subsequent separation and analysis of phosphorylated peptides and palmitoylated peptides.

[0039] Comparative Example 3 The experimental steps and conditions were the same as those in Example 1, except that in step (1), 0.6 g Ce(NO3)3·6H2O and 0.9 g urea were added to the mixture, and after stirring and mixing, the mixture was transferred to a 100 mL reactor and reacted at 160°C for 8 h.

[0040] Comparative Example 4 The experimental steps and conditions were the same as those in Example 1, except that in step (1), 0.6 g Ce(NO3)3·6H2O and 0.9 g urea were added to the mixture, stirred and mixed until uniform, and then transferred to a 100 mL reactor and reacted at 120°C for 6 h.

[0041] Figure 4 The scanning electron microscope images of the MCeO2 materials prepared in Comparative Examples 3 and 4 are shown in FIG. Figure 4 (a) represents comparative example 3, Figure 4 (b) represents Comparative Example 4. It can be seen from the figure that the size of MCeO2 synthesized in Comparative Example 3 is too large due to the high reaction temperature during the synthesis process, which is greater than 400 nm, resulting in the subsequent formation of Janus with a large size, and the specific surface area of ​​the material is reduced, which is not suitable for use as an adsorbent in the field of separation and analysis; and the size of MCeO2 synthesized in Comparative Example 4 is too small due to the shortened time during the synthesis process, which is less than 100 nm, and the smaller size of the magnetic material will cause serious agglomeration, which is also not suitable for the subsequent formation of Janus.

[0042] The Janus MCP and Janus MCP-BVA prepared in Example 1 and Comparative Examples 1-4 were used as magnetic adsorbents to analyze phosphorylated peptide and palmitoylated peptide standard samples in the samples.

[0043] Mass spectrometry conditions: The samples to be tested were analyzed using an AB Sciex 5800 time-of-flight mass spectrometer. 0.5 μL of eluent and 0.5 μL of 2,5-dihydroxybenzoic acid (DHB) were added to the same point on the MALDI-TOF mass spectrometry target plate and mixed evenly. After crystallization, the sample was vacuumed and analyzed. The remaining parameters were set as follows: positive ion mode was selected; acceleration voltage was 20 kV; laser pulse frequency was 400 Hz; mass scanning range was 1000-3500; laser source was Nd-YAG.

[0044] Sample preparation: Phosphorylated peptide standard: weigh 1 mg β -Casein ( β -casein) lyophilized powder standard was dissolved in 1 mL of ammonium bicarbonate buffer solution (50 mM, pH 8.3) and placed in a 100 ℃ metal bath for 10 minutes to allow the protein to undergo thermal denaturation. After the protein solution was cooled to room temperature, 1 mg / mL of trypsin (substrate: enzyme = 30:1, w / w ), enzymatic hydrolysis reaction occurred in a 37 ℃ water bath oscillator, and 2 μL of formic acid was added to terminate the enzymatic hydrolysis reaction after 16 hours. The obtained standard sample solution was diluted with solution 1 (50% acetonitrile + 0.1% trifluoroacetic acid). Before each test, the sample solution was diluted to a concentration of 50 pmol for subsequent analysis and testing; Palmitoylated peptide standard: Use the self-synthesized palmitoylated peptide (synthesis method reference HJ Zheng et al. Simultaneous Profiling of Palmitoylomics and Glycomics with Photo / pHDual-Responsive Magnetic Nanocomposites. Small Methods 2023, 2300254.). Before each test, dilute the sample solution to a concentration of 50 pmol for subsequent analysis and testing; Enrichment process: 20 μg of Janus MCP and Janus MCP-BVA magnetic adsorbents were weighed respectively, and washed repeatedly with ultrapure water and solution 1 for 5 times respectively. Then, the magnetic adsorbents were dispersed in 1 mL of solution 1 as a reserve solution for later use.

[0045] The prepared magnetic adsorbent was used in magnetic solid phase microextraction (MSPE) technology, and the steps were loading, washing, and desorption; To enrich phosphorylated peptides, 20 μL of 20 μg / mL Janus MCP and Janus MCP-BVA magnetic adsorbents were added to 100 μL of buffer (50% acetonitrile + 0.1% trifluoroacetic acid, pH 3.5) containing phosphorylated peptides and shaken at room temperature for 30 min. The material was separated by magnetic absorption and washed three times with the above buffer, and then dissolved in 30 μL 1% NH3·H2O to elute the phosphorylated peptides for subsequent mass spectrometry analysis. The mass spectra of the Janus MCP and Janus MCP-BVA materials prepared in Example 1 for detecting phosphorylated peptides are shown in Figure 1. Figure 5 (a, b, c) show that the mass spectra of the samples before enrichment are as follows Figure 5 (a), Figure 5 (b) and Figure 5 (c) are the mass spectra of phosphorylated peptides enriched by Janus MCP and Janus MCP-BVA. After enrichment, 6 phosphorylated peptide signal peaks were obtained (mass-to-charge ratios were 1481.8, 1660.3, 1832.3, 2061.5, 2556.8, and 3122.4, respectively), indicating that the synthesized Janus MCP and Janus MCP-BVA have good separation and enrichment capabilities for phosphorylated peptides.

[0046] To enrich palmitoylated peptides, 20 μL of 20 μg / mL Janus MCP and Janus MCP-BVA magnetic adsorbents were added to 100 μL of palmitoylated peptide buffer (50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 5.5) and shaken at room temperature for 30 min. The material was separated by magnetic absorption and washed three times with the above buffer, and then the material was dissolved in 50% acetonitrile + 0.1% trifluoroacetic acid to elute the palmitoylated peptides for subsequent mass spectrometry analysis. Example 1 Detection of palmitoylated peptides by Janus MCP and Janus MCP-BVA materials prepared as Figure 5 As shown in (d, e, f), the mass spectra of the samples before enrichment are as follows Figure 5 (d), while Figure 5 (e) and Figure 5 (f) Mass spectra of phosphorylated peptides enriched by Janus MCP and Janus MCP-BVA magnetic adsorbents. Figure 5 As shown in (e), after the sample was enriched by Janus MCP, no signal peak of palmitoylated peptide was generated, indicating that the synthesized Janus MCP only has the ability to separate and enrich phosphorylated peptides, but not palmitoylated peptides. Figure 5As shown in (f), a signal peak of palmitoylated peptide with a strong signal value (mass-to-charge ratio of 1005.9) is generated, indicating that Janus MCP-BVA has a strong separation and enrichment ability for both phosphorylated peptides and palmitoylated peptides. Specific data of Example 1 and Comparative Examples 1-4 are shown in Table 1.

[0047] Table 1

[0048] The experimental results show that the Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications synthesized under the material ratio and operating conditions of Example 1 has the best enrichment effect on phosphorylated peptides and palmitoylated peptides in the sample.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications, characterized in that: The steps include: Step 1: Prepare CeO2-coated ferroferric oxide magnetic nanoparticles MCeO2; Step 2: Mix the MCeO2, hexadecyltrimethylammonium bromide and ammonia water obtained in step 1 to obtain a mixed solution, add 1,2-bis(triethoxysilane)ethane to the mixed solution for reaction to obtain a product, disperse the product in NH4NO3 solution for reflux reaction to obtain a Janus material composed of mesoporous silicon in situ grown on magnetic CeO2, referred to as Janus MCP; Step 3: The Janus MCP obtained in step 2 is reacted with a silane coupling agent to obtain an amino-modified Janus MCP. The amino-modified Janus MCP and 4-ethylenesulfonylbenzoic acid are dissolved in a buffer solution containing EDC and NHS to react to obtain a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications.

2. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The size of the CeO2-coated ferroferric oxide magnetic nanospheres MCeO2 described in step 1 is between 200 and 250 nm.

3. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The mass mg of MCeO2 in step 2: the mass mg of hexadecyltrimethylammonium bromide: the volume mL of ammonia water: the mass mg of 1,2-bis(triethoxysilane)ethane are 90:150:1.8:

100.

4. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The reaction temperature for adding 1,2-bis(triethoxysilane)ethane to react in step 2 is room temperature, the rotation speed is 800 rpm, and the reaction time is 3 h.

5. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The reaction temperature of the reflux reaction in step 2 is 80-100° C., and the reaction time is 8-14 h.

6. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The concentration of the NH4NO3 solution in step 2 is 5.0~7.0 g / L.

7. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: In step three, the silane coupling agent is 3-aminopropyltriethoxysilane.

8. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The reaction temperature of the step 3 neutralization silane coupling agent reaction is 50-65° C., and the reaction time is 20-24 h.

9. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: In step 3, the mass ratio of the aminated Janus MCP, 4-ethylenesulfonylbenzoic acid, EDC and NHS is 13.5:3:60:

80.

10. The method for preparing a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification according to claim 1, characterized in that: The reaction temperature in step 3 is room temperature, and the reaction time is 8 to 14 hours.

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