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

Abstract

The present invention provides a preparation method of Janus materials for simultaneous screening of protein phosphorylation and palmitoylation modifications, belonging to the technical field of magnetic material synthesis. This method first prepares MCeO2; MCeO2, cetyltrimethylammonium bromide and ammonia water are mixed to obtain a mixed solution, and 1,2-bis(triethoxysilyl)ethane is added to the mixed solution for reaction to obtain a product, and this product is dispersed in an NH4NO3 solution for reflux reaction to obtain Janus MCP; Janus MCP is reacted with a silane coupling agent, and then the amino-functionalized Janus MCP and 4-vinylsulfonylbenzoic acid are dissolved in a buffer solution containing EDC and NHS for reaction to obtain Janus materials. This preparation method is reliable, the material properties are stable, and the enrichment efficiency of magnetic Janus for target peptide segments (phosphorylated peptide segments and palmitoylated peptide segments) is significantly improved.

Description

Technical Field

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

[0002] Janus nanomaterials have anisotropy in physical and chemical properties and have broadened the application fields with various functionalized modifications, attracting much attention.

[0003] Phosphorylation modification cannot be ignored in proteomics research. Abnormal phosphorylated peptides are often used to distinguish and identify biomarkers related to diseases. 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, which plays an extremely important role in a wide range of biological processes such as cell signal transduction, apoptosis, and carcinogenesis.

[0004] Currently, commonly used enrichment methods for phosphorylation modification include anion exchange chromatography, immobilized metal ion affinity chromatography, metal oxide / hydroxide affinity chromatography, and molecular imprinting, etc. Ideal enrichment materials should have good properties such as specific binding to phosphorylated peptides, easy modification, and convenient elution and separation. Research methods include "palmitate-centered", "cysteine-centered", bioinformatics prediction, and direct mass spectrometry detection methods, etc. However, due to the low abundance and stoichiometric levels of post-modified peptide segments in vivo, the lack of a clear consensus sequence, the absence of available commercial antibodies, and the severe signal suppression of a large number of unmodified peptide segments in mass spectrometry analysis, the idea of completely characterizing protein phosphorylation and palmitoylation modifications still faces great analytical challenges. In particular, there is a great lack of methods for simultaneously screening two important post-modified peptide segments. In view of this, before mass spectrometry analysis, developing a precise and efficient selective enrichment method based on chemical strategies has become a prerequisite for exploring phosphoproteomics and palmitoproteomics.

[0005] Currently, no Janus materials have been developed for simultaneous screening of protein phosphorylation and palmitoylation modifications. Summary of the Invention

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

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A preparation method of a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications, comprising the following steps:

[0009] Step 1: Prepare CeO 2 -coated magnetite magnetic nanospheres MCeO 2 ;

[0010] Step 2: Mix the MCeO 2 obtained in Step 1, cetyltrimethylammonium bromide, and ammonia water to obtain a mixed solution. Add 1,2-bis(triethoxysilyl)ethane to the mixed solution for reaction to obtain a product. Disperse the product in NH 4 NO 3 solution for reflux reaction to obtain a Janus material formed by in-situ growth of mesoporous silica on magnetic CeO 2 , abbreviated as Janus MCP; Janus MCP;

[0011] Step 3: React the Janus MCP obtained in Step 2 with a silane coupling agent to obtain an amino-functionalized Janus MCP. Dissolve the amino-functionalized Janus MCP and 4-vinylsulfonylbenzoic acid in a buffer solution containing EDC and NHS for reaction to obtain a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications.

[0012] Preferably, the size of the CeO 2 -coated magnetite magnetic nanospheres MCeO 2 in Step 1 is between 200 and 250 nm.

[0013] Preferably, the mass mg of MCeO 2 in Step 2: the mass mg of cetyltrimethylammonium bromide: the volume mL of ammonia water: the mass mg of 1,2-bis(triethoxysilyl)ethane is 90:150:1.8:100.

[0014] Preferably, the reaction temperature for adding 1,2-bis(triethoxysilyl)ethane for reaction in Step 2 is room temperature, the rotation speed is 800 rpm, and the reaction time is 3 h.

[0015] Preferably, the reaction temperature for the reflux reaction in Step 2 is 80-100 °C, and the reaction time is 8-14 h.

[0016] Preferably, the concentration of the NH 4 NO 3 solution in Step 2 is 5.0-7.0 g / L.

[0017] Preferably, in step three, the silane coupling agent is 3-aminopropyltriethoxysilane (APTES).

[0018] Preferably, in step three, the reaction temperature for reacting with the silane coupling agent is 50-65 °C, and the reaction time is 20-24 h.

[0019] Preferably, in step three, the mass ratio of the aminated Janus MCP, 4-vinylsulfonylbenzoic acid, carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 13.5:3:60:80.

[0020] Preferably, in step three, the reaction temperature is room temperature, and the reaction time is 8-14 h.

[0021] Advantages of the present invention

[0022] The present invention provides a preparation method of a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modification. The preparation of the Janus material is to first synthesize CeO 2 -coated magnetic iron oxide nanoparticles, and then use a template agent to control the nucleation and growth of periodic mesoporous silica (PMO) on the MCeO 2 material to form Janus MCP. Finally, 4-vinylsulfonylbenzoic acid is modified onto the PMO unit of Janus MCP by a chemical cross-linking method to prepare the final Janus material. The Janus material of the present invention consists of two parts, a magnetic unit and a mesoporous silica unit. A metal oxide CeO 2 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 silica unit, a vinylsulfonyl group with affinity for palmitoylated peptides is modified by utilizing the unique structure of mesoporous silica with multiple modification sites. The preparation method of 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. Using the magnetic adsorbent synthesized in the present invention as the matrix of magnetic solid-phase microextraction (MSPE) technology is applicable to the analysis of phosphorylated peptides and palmitoylated peptides in complex biological samples (such as cell lysates), which proves its excellent application potential in the separation and MSPE fields of proteomics. Brief description of the drawings

[0023] Figure 1 Infrared spectra of the MCeO 2 and Janus MCP materials prepared in Example 1 of the present invention.

[0024] Figure 2 Infrared spectra of the MCeO 2Zeta potential diagram of Janus MCP material.

[0025] Figure 3 Scanning electron microscope images of Janus MCP-BVA materials prepared in Example 1 and Comparative Examples 1-2 of the present invention.

[0026] Figure 4 Scanning electron microscope images of MCeO prepared in Comparative Examples 3 and 4 of the present invention. 2

[0027] Figure 5 Mass spectrometry images of phosphorylated peptides and palmitoylated peptides detected by Janus MCP and Janus MCP-BVA materials prepared in Example 1 of the present invention, respectively. Detailed implementation mode

[0028] A preparation method of a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications, comprising the following steps:

[0029] Step 1: Prepare CeO 2 -coated iron oxide magnetic nanospheres MCeO 2 ;

[0030] Step 2: Mix the MCeO obtained in Step 1 2 , cetyltrimethylammonium bromide and ammonia water to obtain a mixed solution, add 1,2-bis(triethoxysilyl)ethane to the mixed solution for reaction to obtain a product, and disperse the product in NH 4 NO 3 solution for reflux reaction to obtain a Janus material in which mesoporous silica grows in situ on magnetic CeO 2 formed Janus material, abbreviated as Janus MCP;

[0031] Step 3: React the Janus MCP obtained in Step 2 with a silane coupling agent to obtain an amino-functionalized Janus MCP, and dissolve the amino-functionalized Janus MCP and 4-vinylsulfonylbenzoic acid in a buffer solution containing EDC and NHS for reaction to obtain a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications.

[0032] According to the present invention, the preparation method of the CeO 2 -coated iron oxide magnetic nanospheres MCeO 2 preferably includes:

[0033] Step (1-1), weigh FeCl 3 ·6H 2O and sodium acetate are dissolved in a solvent, and the solvent is preferably ethylene glycol. After being uniformly dispersed, a reaction solution is obtained. Then, the reaction solution is added to a reaction kettle for reaction. The reaction temperature is preferably 180 °C, and the reaction time is preferably 14 h to obtain a product. The obtained product is preferably washed alternately with ultrapure water and absolute ethanol, and dried to obtain Fe 3 O 4 nanoparticles. The molar ratio of FeCl 3 ·6H 2 O to sodium acetate is preferably 24:79.5;

[0034] Step (1-2): Weigh the above-mentioned Fe 3 O 4 nanoparticles and dissolve them in a mixed solution containing ethanol, water, and ammonia water. Ultrasonic treatment is performed to make them uniformly dispersed. The ultrasonic time is preferably 30 min. Tetraethyl orthosilicate (TEOS) is slowly added to the above solution for reaction. The reaction temperature is preferably room temperature, and the reaction time is preferably 10 h to obtain Fe 3 O 4 @SiO 2 ; The mass mg of the Fe 3 O 4 nanoparticles: the volume mL of ethanol: the volume mL of water: the volume mL of ammonia water: the volume mL of tetraethyl orthosilicate is preferably 175:110:28:0.7:0.5;

[0035] Step (1-3): Take the above-mentioned Fe 3 O 4 @SiO 2 and disperse it in ultrapure water. Perform ultrasonic treatment. The ultrasonic treatment time is preferably 30 min. Then, Ce(NO 3 ) 3 ·6H 2 O and urea are added to the above mixture. After stirring and mixing, it is added to a reaction kettle for reaction. The reaction temperature is preferably 120 °C, and the reaction time is preferably 8 h. Then, it is separated by the magnetic attraction method, and the brownish-yellow solid is washed repeatedly with ethanol and water, and dried in vacuum to obtain the magnetic material coated with CeO 2 , named MCeO 2 . The mass ratio of the Fe 3 O 4 @SiO 2 , Ce(NO 3 ) 3 ·6H 2 O to urea is preferably 3:6:9. The size of the MCeO 2 is between 200 and 250 nm; In the present invention, by adjusting the reaction temperature and reaction duration in this step, MCeO 2Materials are prepared for subsequent synthesis of Janus materials. When the reaction temperature is too high, the synthesized MCeO 2 has too large a size and is not suitable for subsequent growth into Janus. Similarly, when the reaction time is less than 8 h, the synthesized MCeO 2 has too small a size and is also not suitable for subsequent growth into Janus.

[0036] According to the present invention, the MCeO 2 , cetyltrimethylammonium bromide and ammonia water are respectively added to ultrapure water, and mixed and stirred at room temperature to make them evenly dispersed. The mixing time is preferably 30 min to obtain a mixed solution. 1,2-bis(triethoxysilyl)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, and the reaction time is preferably 3 h. After the reaction stops, it is washed and dried, and the waste solvent is discarded while the product is retained; the present invention obtains Janus materials with appropriate size by adjusting the rotation speed and reaction duration in this step. When the rotation speed is too low, the mesoporous silicon unit in the synthesized Janus material has too small a size and cannot form a Janus with relatively consistent symmetry. When the reaction time is too long, the mesoporous silicon unit in the synthesized Janus material is too long and also cannot form a Janus with good symmetry. 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.

[0037] According to the present invention, the above product is dispersed in NH 4 NO 3 solution for reflux reaction, and after post-treatment, Janus MCP is obtained; the reaction temperature is preferably 80-100 °C, more preferably 90 °C, the reaction time is preferably 8-14 h, more preferably 12 h, and the NH 4 NO 3 solution is preferably an ethanol solution of NH 4 NO 3 , and the concentration of the NH 4 NO 3 solution is preferably 5.0-7.0 g / L, more preferably 6.0 g / L. The post-treatment is preferably: after the above reflux solution is cooled, centrifuged (11000 rpm) to remove the supernatant, and the obtained orange-yellow solid is alternately washed with ethanol and ultrapure water, and the obtained product is vacuum dried at 60 °C for standby. The mass mg of the MCeO 2 : the mass mg of cetyltrimethylammonium bromide: the volume mL of ammonia water: the mass mg of 1,2-bis(triethoxysilyl)ethane: NH 4 NO 3The volume of the solution in mL is preferably 90:150:1.8:100:150.

[0038] According to the present invention, it is preferred to first disperse the obtained Janus MCP in a solvent. The solvent is preferably ethanol to obtain a homogeneous solution. The concentration of the solution is preferably 2 mg / mL. Then, a silane coupling agent is added to the above solution for reaction. The reaction temperature is preferably 50 - 65°C, and the reaction time is preferably 20 - 24 h. After stopping the reaction, after magnetic separation and washing operations, the obtained product is dried to obtain amino-functionalized Janus MCP; the silane coupling agent is preferably 3-aminopropyltriethoxysilane (APTES).

[0039] According to the present invention, EDC (1-ethyl-3-(3-dimethylaminopropyl)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. Then, amino-functionalized Janus MCP and 4-vinylsulfonylbenzoic acid (BVA) are dissolved in the buffer solution containing EDC and NHS for reaction. The reaction temperature is preferably room temperature, and the reaction time is preferably 8 - 14 h, more preferably 12 h. 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 amino-functionalized Janus MCP, 4-vinylsulfonylbenzoic acid, EDC, and NHS is preferably 13.5:3:60:80.

[0040] The present invention selects Fe 3 O 4 magnetic nanoparticles as one side of the Janus structure, as a carrier for magnetic solid-phase extraction to perform pretreatment on samples in proteomics, facilitating elution while improving the enrichment ability; the other side is periodic mesoporous silica (PMO), and the porous structure provides abundant binding sites. BVA is modified onto PMO through an NHS-EDC coupling reaction, and CeO 2 is coated onto the magnetic beads by in-situ precipitation. Combining MALDI-TOF-MS mass spectrometry analysis, dual enrichment of phosphorylated peptides and palmitoylated peptides is achieved.

[0041] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The raw materials involved in the embodiments are all commercially available. Example 1

[0042] A preparation method of a Janus material for simultaneous screening of protein phosphorylation and palmitoylation modifications, comprising the following steps:

[0043] Step (1), synthesize CeO 2 -coated magnetic iron oxide nanoparticles (named MCeO 2 ):

[0044] Weigh 3.24 g of FeCl 3 ·6H 2 O (24 mmol) and 6.52 g of sodium acetate (79.5 mmol), dissolve them in 120 mL of ethylene glycol, and disperse them evenly. Take 60 mL of the above solution and transfer it to a 100 mL polytetrafluoroethylene (PTFE) reactor, react in an oven at 180 °C for 14 h, after cooling to room temperature, separate the obtained magnetic iron oxide nanoparticles (Fe 3 O 4 ) by magnetic separation method, wash them alternately with ultrapure water and absolute ethanol 5 times each, and then dry them in a vacuum oven at 60 °C for standby. Weigh 175 mg of Fe 3 O 4 nanoparticles and dissolve them in a mixed solution containing 110 mL of ethanol, 28 mL of water and 0.7 mL of ammonia water, ultrasonically disperse them for 30 min, slowly add 0.5 mL of tetraethyl orthosilicate (TEOS), stir at room temperature for 10 h, after the reaction stops, wash and dry them by the above method to obtain Fe 3 O 4 @SiO 2 . Take 300 mg of Fe 3 O 4 @SiO 2 disperse it in 120 mL of ultrapure water, ultrasonically treat it for 30 min, then add 0.6 g of Ce(NO 3 ) 3 ·6H 2 O and 0.9 g of urea to the mixture, stir and mix evenly, then transfer it to a 100 mL reactor, react at 120 °C for 8 h, separate it by magnetic separation method, and then repeatedly wash the yellowish-brown solid with ethanol and water, and vacuum dry it to obtain the CeO 2 -coated magnetic material, named MCeO 2 .

[0045] Step (2), use a template agent to control the nucleation and growth of periodic mesoporous silica (PMO) on the MCeO 2 material under alkaline conditions to form a Janus material (named Janus MCP):

[0046] Take 90 mg of MCeO2 , 150 mg of cetyltrimethylammonium bromide (CTAB) and 1.8 mL of ammonia water were added to 100 mL of ultrapure water, and stirred at room temperature for 30 min to disperse evenly. 1,2-Bis(triethoxysilyl)ethane (BTEE) was added dropwise to the mixture, the rotation speed was adjusted to 800 rpm, and the reaction was carried out at room temperature for 3 h. After the reaction stopped, it was washed and dried, the waste solvent was discarded, and the product was retained. The product was dispersed in 150 mL of an ethanol solution containing 6.0 g / L of NH 4 NO 3 , and refluxed at 90 °C overnight. After the above reflux solution cooled, the supernatant was removed by centrifugation (11,000 rpm), and the obtained orange-yellow solid was washed alternately with ethanol and ultrapure water. The obtained product was dried in vacuo at 60 °C for standby, and named Janus MCP.

[0047] Step (3), using the chemical cross-linking method, 4-vinylsulfonylbenzoic acid (BVA) was modified onto the PMO unit of Janus MCP (named Janus MCP-BVA):

[0048] The Janus MCP material was dispersed in ethanol to form a uniform solution of 2 mg / mL. 50 mL of the solution was taken, 0.2 mL of silane coupling agent (ATPES) was added, and the reaction was carried out at 50 °C for 24 h. After the reaction stopped, after magnetic separation and washing operations, the obtained product was dried in a vacuum oven at 60 °C to obtain amino-functionalized 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-functionalized Janus MCP and 3 mg of BVA were added to the above buffer solution, and stirred overnight in the dark at room temperature. After the reaction stopped, after magnetic separation and washing operations, the obtained product was dried in a vacuum oven at 60 °C to obtain the final product, named Janus MCP-BVA.

[0049] Figure 1 For the MCeO prepared in Example 1 of the present invention 2 and the infrared spectra of the Janus MCP material, where curve (a) represents MCeO 2 , curve (b) represents the Janus MCP material. In curve (a), the peak at ~580 cm -1 is attributed to the lattice vibration of Fe 3 O 4 ; the characteristic peaks generated at 1075 and 845 cm -1 correspond to the symmetric and antisymmetric stretching vibrations of Si-O-Si; at 1506 and 1411 cm -1The two peaks at [specific wavenumber] are related to the bending vibration of the hydroxyl groups on cerium oxide (Ce-OH); the peak at 3450 cm -1 corresponds to the stretching vibration of O-H in water, indicating the successful synthesis of MCeO 2 . In curve (b), the peak at ~580 cm -1 still belongs to the lattice vibration of Fe 3 O 4 . The peak values of the two Ce-OH peaks at 1506 and 1411 cm -1 decrease, proving that part of the surface of MCeO 2 is covered. The two new peaks at 1041 and 916 cm -1 , representing the Si-C peak and the Si-O-Si peak of linear polysiloxane, prove the successful synthesis of Janus MCP.

[0050] Figure 2 Figure [figure number] shows the ZETA potential diagrams of the MCeO 2 and Janus MCP materials prepared in Example 1 of the present invention. Figure 2 As shown, due to coating CeO 2 on the surface of magnetic spheres under alkaline conditions, the carried hydroxyl groups result in a negative potential; during the growth of mesoporous silicon, the positively charged template cetyltrimethylammonium bromide is adsorbed on the surface of the particles through electrostatic interaction, making the potential become positive. The obtained results are consistent with the prediction, proving the successful synthesis of MCeO 2 and Janus MCP materials.

[0051] Comparative Example 1

[0052] The experimental procedures and conditions are the same as those in Example 1, except that the rotation speed in step (2) is adjusted to 200 rpm and other conditions remain unchanged.

[0053] Comparative Example 2

[0054] The experimental procedures and conditions are the same as those in Example 1, except that the reaction duration in step (2) is adjusted to react for 6 h at room temperature and other conditions remain unchanged.

[0055] Figure 3 Figure [figure number] shows the scanning electron microscope images of the Janus MCP-BVA materials prepared in Example 1 of the present invention and Comparative Examples 1-2, where Figure 3 (a) represents Comparative Example 2, Figure 3 (b) represents Comparative Example 1, Figure 3(c) represents Example 1. As can be seen from the figure, during the synthesis of Janus in Comparative Example 2, due to the too long reaction duration, the growth size of the mesoporous silicon part of Janus is too long, with a size greater than 1 μm, which is not suitable for the separation and analysis of target analytes; while during the synthesis of Janus in Comparative Example 1, due to the too slow rotation speed, the growth size of the mesoporous silicon part of Janus is small. As can be seen from Figure 3 (b), the formed Janus structure is significantly asymmetric. And due to the slow reaction rotation speed, the magnetic materials agglomerate seriously, and the formed Janus is also not suitable for the separation and analysis of target analytes; while during the synthesis of Janus in Example 1 of the present invention, both the reaction rotation speed and the reaction duration are well optimized. As can be seen from Figure 3 (c), the formed Janus has a uniform morphology and good symmetry. The Janus magnetic adsorbent obtained under this reaction condition is very suitable for the subsequent separation and analysis of phosphorylated peptides and palmitoylated peptides.

[0056] Comparative Example 3

[0057] The experimental steps and conditions are the same as those in Example 1, except that in step (1), 0.6 g of Ce(NO 3 ) 3 ·6H 2 O and 0.9 g of urea are added to the mixture. After stirring and mixing evenly, it is transferred to a 100 mL reaction kettle and reacted at 160 °C for 8 h.

[0058] Comparative Example 4

[0059] The experimental steps and conditions are the same as those in Example 1, except that in step (1), 0.6 g of Ce(NO 3 ) 3 ·6H 2 O and 0.9 g of urea are added to the mixture. After stirring and mixing evenly, it is transferred to a 100 mL reaction kettle and reacted at 120 °C for 6 h.

[0060] Figure 4 Figures are the scanning electron microscope images of the MCeO 2 materials prepared in Comparative Examples 3 and 4. Among them Figure 4 (a) represents Comparative Example 3, Figure 4 (b) represents Comparative Example 4. As can be seen from the figure, during the synthesis of MCeO 2 in Comparative Example 3, due to the too high reaction temperature, the size of MCeO 2 is too large, with a size greater than 400 nm, which will lead to too large Janus size formed subsequently and the reduction of the specific surface area of the material, and it is not suitable for being used as an adsorbent in the field of separation and analysis; while for the MCeO 2During the synthesis process, due to the shortened duration, the size of MCeO 2 is too small, with a size less than 100 nm. Moreover, magnetic materials with smaller sizes will cause serious agglomeration phenomena and are not suitable for the subsequent formation of Janus either.

[0061] Using the two materials, Janus MCP and Janus MCP-BVA prepared in Example 1 and Comparative Examples 1-4, as magnetic adsorbents, the phosphorylated peptide and palmitoylated peptide standard samples in the sample were analyzed.

[0062] Mass spectrometry conditions: The sample to be measured was analyzed using an AB Sciex 5800 type time-of-flight mass spectrometer. Take 0.5 μL of the eluate and 0.5 μL of 2,5-dihydroxybenzoic acid (DHB) and drop them at the same point on the MALDI-TOF mass spectrometry target plate and mix evenly. After crystallization, vacuum injection was carried out for analysis. The remaining parameter settings are as follows: Cation mode was selected; the acceleration voltage was 20 kV; the laser pulse frequency was 400 Hz; the mass scanning range was 1000 - 3500; the laser source was Nd-YAG.

[0063] Sample preparation: Phosphorylated peptide standard: Weigh 1 mg of β -casein β freeze-dried powder standard, dissolve it in 1 mL of ammonium bicarbonate buffer solution (50 mM, pH 8.3), and let the protein undergo a thermal denaturation reaction in a 100 °C metal bath for 10 minutes. After the protein solution cooled to room temperature, add trypsin at a ratio of 1 mg / mL (substrate: enzyme = 30:1, w / w ), and let the enzymatic hydrolysis reaction occur in a 37 °C water bath oscillation device. After 16 hours, add 2 μL of formic acid to terminate the enzymatic hydrolysis reaction. 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 sample solution with a concentration of 50 pmol for subsequent analysis and testing;

[0064] Palmitoylated peptide standard: Use the synthesized palmitoylated peptide segment (synthesis method: refer to HJ Zheng etal. Simultaneous Profiling of Palmitoylomics and Glycomics with Photo / pHDual-Responsive Magnetic Nanocomposites. Small Methods 2023, 2300254.). Before each test, the sample solution was diluted to a sample solution with a concentration of 50 pmol for subsequent analysis and testing;

[0065] Enrichment process: Weigh 20 μg of each of the two magnetic adsorbents, Janus MCP and Janus MCP-BVA, and wash them 5 times repeatedly with ultrapure water and Solution 1. Subsequently, disperse the magnetic adsorbents in 1 mL of Solution 1 as a stock solution for later use.

[0066] Apply the prepared magnetic adsorbents to the magnetic solid-phase microextraction (MSPE) technique, and the steps are successively loading, washing, and desorbing.

[0067] Enrich phosphopeptides. Add 20 μL of each of the two magnetic adsorbents, Janus MCP and Janus MCP-BVA, at 20 μg / mL to 100 μL of a buffer solution containing phosphopeptides (50% acetonitrile + 0.1% trifluoroacetic acid, pH 3.5), and oscillate at room temperature for 30 min. Separate by magnetic attraction and wash the material three times with the above buffer solution. Then dissolve the material in 30 μL of 1% NH 3 ·H 2 O to elute the phosphopeptides for subsequent mass spectrometry analysis. The mass spectrometry diagrams of the Janus MCP and Janus MCP-BVA materials prepared in Example 1 for detecting phosphopeptides are as shown in Figure 5 (a, b, c). The mass spectrometry diagrams of the samples before enrichment are as shown in Figure 5 (a), Figure 5 (b), and Figure 5 (c) are the mass spectrometry diagrams of enriching phosphopeptides using the two magnetic adsorbents, Janus MCP and Janus MCP-BVA, respectively. After enrichment, 6 signal peaks of phosphopeptides are obtained (mass-to-charge ratios are 1481.8, 1660.3, 1832.3, 2061.5, 2556.8, 3122.4), indicating that the synthesized Janus MCP and Janus MCP-BVA have good separation and enrichment capabilities for phosphopeptides.

[0068] Enrich palmitoylated peptides. Add 20 μL of each of the two magnetic adsorbents, Janus MCP and Janus MCP-BVA, at 20 μg / mL to 100 μL of a buffer solution containing palmitoyl peptides (50 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 5.5), and oscillate at room temperature for 30 min. Separate by magnetic attraction and wash the material three times with the above buffer solution. Then dissolve the material in 50% acetonitrile + 0.1% trifluoroacetic acid to elute the palmitoylated peptides for subsequent mass spectrometry analysis. The detection of palmitoylated peptides by the Janus MCP and Janus MCP-BVA materials prepared in Example 1 is as shown in Figure 5 (d, e, f). The mass spectrometry diagrams of the samples before enrichment are as shown in Figure 5 (d), while Figure 5 (e), and Figure 5(f) are the mass spectrometry diagrams of enriching phosphorylated peptides using two magnetic adsorbents, Janus MCP and Janus MCP-BVA. As Figure 5 shown in (e), after the sample was enriched by Janus MCP, no signal peak of palmitoylated peptides was generated, indicating that the synthesized Janus MCP only has the ability to separate and enrich phosphorylated peptides and has no ability to separate and enrich palmitoylated peptides. After the sample was enriched by Janus MCP-BVA, as Figure 5 shown in (f), a signal peak of palmitoylated peptides with a relatively strong signal value (mass-to-charge ratio of 1005.9) was generated, indicating that Janus MCP-BVA has a strong ability to separate and enrich both phosphorylated peptides and palmitoylated peptides. The specific data of Example 1 and Comparative Examples 1-4 are shown in Table 1.

[0069] Table 1

[0070]

[0071] The experimental results show that the Janus material synthesized under the material ratio and operating conditions of Example 1 for simultaneous screening of protein phosphorylation and palmitoylation modification has the best enrichment effect on phosphorylated peptides and palmitoylated peptides in the sample.

[0072] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall 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 ferroferric oxide magnetic nano-microspheres MCeO2 coated with CeO2 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 for reaction 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 neutralization reaction of the silane coupling agent in step 3 is 50-65° C., and the reaction time is 20-24 hours.

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: In step 3, the aminated Janus MCP and 4-ethylenesulfonylbenzoic acid are dissolved in a buffer solution containing EDC and NHS to carry out a reaction at room temperature for 8 to 14 hours.

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