Preparation method and application of chondroitin sulfate modified mesoporous silica material

Abstract

The application discloses a kind of mesoporous silica as base, natural biomolecule chondroitin sulfate as modified phase functional composite material.The preparation method of the material is disclosed simultaneously, with Glymo silane reagent as connecting arm, polyethylene imine is grafted to the surface of mesoporous silica, then by polyethylene imine and chondroitin sulfate Michael addition reaction obtains chondroitin sulfate modified mesoporous silica material (MSN-PEI-CS).Material is determined by TEM, and with human serum immunoglobulin G as model sample, the selective enrichment effect of material to glycopeptide is explored.The experimental results show that the material shows good detection limit, enrichment capacity and enrichment selectivity.The material is post-modified to introduce Ti 4+ MSN-PEI-CS-PO3 2‑ -Ti 4+ , glycopeptide and phosphorylated peptide can be co-enriched.The MSN-PEI-CS and derived material MSN-PEI-CS-PO3 2‑ -Ti 4+ of the application have wide application prospect in post-translational modification proteomics, and provide new ideas for material design.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation of chondroitin sulfate modified mesoporous silica material, and its enrichment and separation of glycopeptides in a hydrophilic mode; and co-enrichment of glycopeptides and phosphorylated peptides by post-modification of the material. BACKGROUND

[0002] Protein glycosylation and phosphorylation, as an important type of post-translational modification of proteins, are closely related to many life activities. Abnormal expression of protein glycosylation and phosphorylation can cause the occurrence of certain cancers. There are a relatively large number of biomarkers in clinic derived from glycoproteins and phosphorylated proteins. Therefore, accurate identification of abnormal glycosylation and phosphorylation sites has potential application value for early monitoring of diseases and discovery of biomarkers (Ohtsubo K., Marth J D. Cell . 2006, 126(5): 855-867.). However, the low abundance of glycopeptides and phosphorylated peptides in vivo, the interference of non-modified peptides, and the low ionization efficiency will affect the sensitivity and accuracy of biological mass spectrometry. Therefore, it is particularly important to develop new and efficient functional composite materials for enrichment and separation of glycopeptides and phosphorylated peptides before mass spectrometry. At present, the main methods for enrichment of glycopeptides include lectin affinity chromatography, hydrazide chemistry, boron affinity chromatography, and hydrophilic interaction chromatography (Ongay S., Boichenko A., Govorukhina N., Bischoff R. J. Sep. Sci. 2012, 35(18): 2341-2372). The main methods for enrichment of phosphorylated peptides include immunoprecipitation, chemical derivatization, ion exchange chromatography, immobilized metal ion affinity chromatography, and metal oxide affinity chromatography (Li P Z., Wang Y P. Prog. Chem. 2012, 24 (9): 1785-1793).

[0003] Chondroitin sulfate is a natural glycosaminoglycan with various anti-inflammatory, antithrombotic, anticoagulant, antioxidant and other biological activities. So far, it has been widely used in many fields such as biology, biotechnology, food and medicine. Chondroitin sulfate contains a large number of hydroxyl groups on its surface, thus having good hydrophilicity and providing a large number of active sites, which makes it have good application prospect in the development of functional composite materials. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a chondroitin sulfate modified mesoporous silica material. Another technical problem to be solved by the present application is to provide a preparation method of the chondroitin sulfate modified mesoporous silica material. The present application also solves the technical problem of providing the application of the chondroitin sulfate modified mesoporous silica material.

[0005] The technical scheme of the present application is a chondroitin sulfate modified mesoporous silica material, which has the following structure, as shown in the figure: Figure 1

[0006] In the figure, the blue ball represents a polyethyleneimine modified mesoporous silica material (MSN-PEI), and the pore size of the mesoporous silica (MSN) is 2-10 nm; chondroitin sulfate has two subtypes: chondroitin sulfate A and chondroitin sulfate C.

[0007] The mesoporous material has the following characteristics: the NH2 in the polyethyleneimine undergoes an epoxy ring-opening reaction with the GLYMO silane coupling agent; the silicon alcohol group of the GLYMO silane coupling agent can be easily grafted with the -OH in the mesoporous silica; and the chondroitin sulfate is combined with the polyethyleneimine in the MSN-PEI through a Michael addition reaction.

[0008] The technical route of the present application is: mesoporous silica is connected with polyethyleneimine by using a GLYMO silane reagent to obtain a polyethyleneimine modified mesoporous silica (MSN-PEI); and then the MSN-PEI is reacted with chondroitin sulfate to obtain a chondroitin sulfate modified mesoporous silica material (MSN-PEI-CS).

[0009] The present application selects mesoporous silica as the matrix, polyethyleneimine as the connecting arm, and chondroitin sulfate as the modification phase to synthesize the material. The GLYMO silane reagent is connected with the polyethyleneimine through an epoxy ring-opening reaction, and the silicon alcohol group thereon is reacted with the -OH in the mesoporous silica to be connected to the surface of the mesoporous silica, so as to realize the grafting of the polyethyleneimine on the surface of the mesoporous silica. Then, a Michael addition reaction occurs to obtain a chondroitin sulfate modified mesoporous silica material (MSN-PEI-CS).

[0010] The present application also provides a preparation method of a chondroitin sulfate modified mesoporous silica material, comprising the following steps:

[0011] a. 1-2 g of chondroitin sulfate powder is dissolved in 2-100 mL of ultrapure water, 10-20 g of methacrylic anhydride is slowly added, and then a sodium hydroxide solution with a mass concentration of 1-40% is added, and the mixture is stirred at room temperature for 1-24 hours and is placed in a 6℃ refrigerator for 4-72 hours;

[0012] b. The product is washed with water for 2-4 times, and is freeze-dried to obtain a chondroitin sulfate precursor CS-MA;

[0013] ​c. Place 1-2 g of hexadecyltrimethylammonium chloride and 0.1-0.5 mL of triethanolamine into a reaction vessel, add 20-50 mL of deionized water, and reflux at 60-100℃ for 1-24 h. Then add 1-5 mL of tetraethyl orthosilicate, and stir and reflux at 60-100℃ for 1-24 h.

[0014] d. Wash the product with water 2-4 times, then wash with organic reagent 2-4 times;

[0015] e. Vacuum drying at 60-100℃ for 12-48 h yields mesoporous silica MSN;

[0016] f. Place 0.2-1 g of Glymo silane reagent and 0.1-2 g of polyethyleneimine (PEI) sequentially into a reaction vessel, and add 50-100 mL of [the solution / material]. N - N Dimethylformamide was stirred and refluxed at 70-100°C for 12-48 h. MSN was then added, and the mixture was stirred and refluxed at 70-100°C for 12-48 h.

[0017] g. Use N - N Wash the product with dimethylformamide 2-4 times, then wash with organic reagents 2-4 times;

[0018] h. Washed several times with water and freeze-dried to obtain polyethyleneimine-modified mesoporous silica MSN-PEI;

[0019] i. Dissolve MSN-PEI and CS-MA separately in the reagents and then mix them together. React at room temperature with stirring for 24-48 h.

[0020] j. Wash the product 2-4 times with dimethyl sulfoxide and 2-4 times with water, then freeze-dry.

[0021] According to a method for preparing a chondroitin sulfate-modified mesoporous silica material of the present invention, preferably, the stirring in step a is magnetic stirring with a rotation speed of 80-100 rpm.

[0022] According to a method for preparing a chondroitin sulfate-modified mesoporous silica material of the present invention, preferably, the product washed with water in step b needs to be precipitated with ethanol and then centrifuged and washed with water.

[0023] According to a method for preparing a chondroitin sulfate-modified mesoporous silica material of the present invention, preferably, the organic solvent in step d is ethanol, methanol, acetonitrile, acetone, etc.

[0024] Preferably, the organic solvent in step g is ethanol, methanol, acetonitrile, acetone or the like.

[0025] Preferably, in the preparation method of the chondroitin sulfate modified mesoporous silica material according to the application, the CS-MA and the MSN-PEI are dissolved in water and dimethyl sulfoxide respectively in step i; the ratio of the amount of the solvent (mL) to the amount of CS-MA (g) is 40-50:1; the ratio of the amount of the solvent (mL) to the amount of MSN-PEI (mg) is 3-4:5; the mass ratio of MSN-PEI to CS-MA is 1-2:10; and the stirring mode is magnetic stirring at a speed of 80-100 rpm.

[0026] The application further provides the use of the chondroitin sulfate modified mesoporous silica material in the specific enrichment of glycopeptides in a hydrophilic mode.

[0027] In the use of the application, the glycopeptides are enriched by using a column solid phase extraction (SPE) mode.

[0028] An appropriate amount of MSN-PEI-CS is loaded in an SPE column, and the material is cleaned and balanced with a sample loading solution.

[0029] The sample loading solution is ACN / H2O / TFA=88-95:4-11:1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid);

[0030] The elution solution is ACN / H2O / TFA=0-1:97-99:1-3, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid).

[0031] The elution solution is ACN / H2O / TFA=0-1:97-99:1-3, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid).

[0032] The application also provides a preparation method of the material obtained by post-modification of the above chondroitin sulfate modified mesoporous silica material, and application in glycopeptide and phosphorylated peptide co-enrichment. 4+ MSN-PEI-CS-PO3 2- -Ti 4+ The hydrophilicity and metal ion affinity of the material make it have excellent application prospects in glycopeptide and phosphorylated peptide co-enrichment.

[0033] The application provides a preparation method of post-modification of the above chondroitin sulfate modified mesoporous silica material:

[0034] a. 50-100 mg of MSN-PEI-CS and 100-500 mg of N, N-carbonyldiimidazole dispersed in 20-100 mL of DMSO are sequentially added, and stirring reaction is carried out at room temperature for 12-48 h. Then 100-500 mg of aminomethylphosphonic acid dispersed in 20-100 mL of DMF is added, and stirring reaction is carried out at room temperature for 1-12 h;

[0035] b. After the reaction is completed, the material is washed with an organic reagent. Freeze-drying is performed to obtain MSN-PEI-CS-PO3 2- ;

[0036] c. The MSN-PEI-CS-PO3 2- is dispersed in 100-200 mL of a Ti(SO4)2 solution, and stirring reaction is carried out at room temperature for 1-12 h. After the reaction is completed, water washing is performed, and freeze-drying is performed.

[0037] According to the preparation method of post-modification of the chondroitin sulfate modified mesoporous silica material, preferably, the stirring in step a is magnetic stirring, and the rotating speed is 80-100 rpm.

[0038] According to the preparation method of post-modification of the chondroitin sulfate modified mesoporous silica material, preferably, the organic reagent in step b is DMSO, DMF, ethanol, methanol or the like.

[0039] According to the preparation method of post-modification of the chondroitin sulfate modified mesoporous silica material, preferably, the concentration of the Ti(SO4)2 solution in step c is 0.1-1 M; and the mass ratio of the MSN-PEI-CS-PO3 2- to titanium sulfate is 1-10:100.

[0040] In the above application of the present application, the centrifugal method is used to co-enrich glycopeptides and phosphorylated peptides. The sample solution mixed with the material is mixed with the protease solution, the non-modified peptides combined with the material are washed out by the washing solution, and finally the modified peptides are eluted by the elution solution. The elution solution is collected for subsequent mass spectrometry analysis.

[0041] An appropriate amount of MSN-PEI-CS-PO3 2- -Ti 4+ After washing and equilibrating the material with the sample solution, a material solution of a certain concentration is prepared. The protease solution is mixed with a certain amount of material solution to ensure the combination of the material and the modified peptides. Then a certain amount of washing solution is used for washing to remove the non-modified peptides combined with the material. Finally, the modified peptides combined with the material are eluted twice with the elution solution. The elution solution is collected, and the enrichment of the modified peptides is analyzed by MALDI-TOF.

[0042] The sample solution is: ACN / H2O / TFA=88-95:4-11:1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid);

[0043] The washing solution is: ACN / H2O / TFA=88-95:4.9-10.9:0.1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid);

[0044] The elution solution is: ACN / H2O / TFA=0-1:97-99:1-3, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid), and 0.4 M NH4OH.

[0045] The present application has the following advantages:

[0046] 1. The synthesis method is simple, and the raw materials are cheap and easy to obtain;

[0047] 2. Selecting mesoporous silica with large specific surface area as the substrate provides more active sites;

[0048] 3. Using chondroitin sulfate, a natural biomolecule, as a linking arm enhances the hydrophilicity of the material;

[0049] 4. The hydrophilicity of chondroitin sulfate-modified mesoporous silica material enables efficient and selective enrichment of glycopeptides;

[0050] 5. Chondroitin sulfate-modified mesoporous silica material chelates Ti 4+ MSN-PEI-CS-PO3 2- -Ti 4+ has good application prospects in co-enrichment of glycopeptides and phosphorylated peptides;

[0051] 6: The glycopeptide and phosphorylated peptide obtained by the method can be directly detected by a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF), thereby improving the detection sensitivity of the mass spectrometer. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 A schematic diagram of the structure of the chondroitin sulfate modified mesoporous silica material.

[0053] Figure 2 A process diagram of the preparation of the chondroitin sulfate modified mesoporous silica material.

[0054] Figure 3 A transmission electron microscope image of the chondroitin sulfate modified mesoporous silica material in Example 1.

[0055] Figure 4 A glycopeptide enrichment image of the chondroitin sulfate modified mesoporous silica material in Example 1.

[0056] Figure 5 A glycopeptide enrichment image of the chondroitin sulfate modified mesoporous silica material in Example 2 using a complex sample (high non-sugar peptide incorporation ratio IgG:BSA = 1:500).

[0057] Figure 6 A glycopeptide and phosphorylated peptide co-enrichment image of MSN-PEI-CS-PO3 2- -Ti 4+ in Example 7. EMBODIMENTS

[0058] The sample loading liquid is composed as shown in a-b:

[0059] a. ACN / H2O / TFA = 90:9:1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid);

[0060] b. ACN / H2O / TFA = 92:7:1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid).

[0061] The elution liquid is composed as shown in a-b:

[0062] ACN / H2O / TFA = 90:9.9:0.1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid).

[0063] The elution liquid is composed as shown in a-b:

[0064] a. ACN / H2O / TFA = 0:99:1, v / v / v (ACN is acetonitrile, H2O is water, and TFA is trifluoroacetic acid);

[0065] b. ACN / H2O / TFA = 0:98:1, v / v / v (ACN is acetonitrile, H2O is water, TFA is trifluoroacetic acid);

[0066] c. 0.4 mol / L ammonia water.

[0067] The concentration of the peptide sample is 1 mg / mL, 5 mg / mL.

[0068] The peptide sample is a proteolysis solution.

[0069] The protein is human serum immunoglobulin G (IgG), standard phosphorylated protein (alpha-casein) or bovine serum albumin (BSA).

[0070] The enzyme is trypsin.

[0071] The collected eluate is detected by a matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF). Embodiment

[0072] Dissolve 1 g of chondroitin sulfate in 50 mL of ultrapure water, then slowly add 10.34 g of methacrylic anhydride, and then slowly add 7.5 mL of 5 N sodium hydroxide solution to undergo methacrylation of chondroitin sulfate. After stirring at room temperature for 2 hours, stand in a refrigerator at 6°C for 24 hours, precipitate by adding ethanol, wash with water, centrifuge, and freeze-dry to obtain chondroitin sulfate precursor (CS-MA). Dissolve 2 g of CTAC in 20 mL of deionized water, slowly add 0.32 mL of TE, and react at 75°C for 2 hours, then slowly add 1.5 mL of TEOS, and react at 75°C for 3 hours. Wash thoroughly with water and ethanol, and dry at 60°C under vacuum to obtain mesoporous silica (MSN). Mix 0.4 g of Glymo silane reagent with 0.5 g of PEI, and then add to 50 mL of DMF, and react at 80°C for 24 hours, then add 2 g of MSN and continue to react for 24 hours. After the reaction is completed, wash thoroughly with DMF and ethanol, and freeze-dry to obtain polyethyleneimine-modified mesoporous silica (MSN-PEI). Dissolve 0.5 g of CS-MA in 20 mL of ultrapure water, mix with 30 mL of DMSO containing 50 mg of MSN-PEI, and stir at room temperature for 48 hours. After the reaction is completed, wash thoroughly with DMSO and water, and freeze-dry to obtain chondroitin sulfate-modified mesoporous silica (MSN-PEI-CS).

[0073] Material characterization: Figure 3 It is a transmission electron micrograph of MSN-PEI-CS. Embodiment

[0074] Take 1 mg MSN-PEI-CS in example 1 to pack in SPE column, wash and balance the material with loading solution. Dissolve 6 μg IgG enzymatic solution in loading solution and slowly push into the SPE column with material, to ensure the full binding of material and glycopeptides (loading solution: ACN / H2O / TFA=92:7.0:1.0, v / v / v). Then wash with 30 μL elution solution for 2 times, to remove the non-glycopeptides combined with the material (elution solution: ACN / H2O / TFA=99:9.9:0.1, v / v / v). Finally, wash with 5 μL elution solution for 2 times to elute the glycopeptides combined with the material (elution solution: ACN / H2O / TFA=0:99:1, v / v / v). Collect the elution solution and analyze the glycopeptide enrichment by MALDI-TOF.

[0075] Glycopeptide enrichment of MSN-PEI-CS is as follows Figure 4 , and the number of enriched glycopeptides is 31. Example

[0076] Take 1.5 mg MSN-PEI-CS in example 1 to pack in SPE column, wash and balance the material with loading solution. Dissolve 6 μg IgG and 265 μg BSA in loading solution and slowly push into the SPE column with material, to ensure the full binding of material and glycopeptides (loading solution: ACN / H2O / TFA=92:7.0:1.0, v / v / v). Then wash with 50 μL elution solution for 10 times, to remove the non-glycopeptides combined with the material (elution solution: ACN / H2O / TFA=99:9.9:0.1, v / v / v). Finally, wash with 5 μL elution solution for 2 times to elute the glycopeptides combined with the material (elution solution: ACN / H2O / TFA=0:99:1, v / v / v). Collect the elution solution and analyze the glycopeptide enrichment by MALDI-TOF.

[0077] Figure 5 Glycopeptide enrichment of MSN-PEI-CS for complex sample (high non-glycopeptide incorporation ratio IgG: BSA=1:500) is as follows, and the number of enriched glycopeptides is 10. Example

[0078] Take 50 mg MSN-PEI-CS and 100 mg CDI to disperse in 20 mL DMSO, and react at room temperature for 24 hours. After the reaction is completed, wash thoroughly with DMSO. Then add 100 mg aminomethyl phosphonic acid to disperse in 20 mL DMF, and react at room temperature for 4 hours. After the reaction is completed, wash thoroughly with DMF. Freeze-drying to obtain MSN-PEI-CS-PO3 2- . Take 50 mg MSN-PEI-CS-PO3 2-MSN-PEI-CS-PO3 2- -Ti 4+ . Example

[0079] MSN-PEI-CS-PO3 2- -Ti 4+ The material was washed and equilibrated twice with 300 μΐ, of loading solution, and then 500 μΐ, of loading solution was added to bring the concentration of the material solution to 2 mg / mL. 6 μg of IgG digest and 2 μg of a-Casein digest were dissolved in 180 μΐ, of loading solution and 20 μΐ, of material solution (containing 40 μg of material) was added. The mixture was sonicated and shaken for 30 min to ensure the material was well combined with the phosphorylated peptides (loading solution: ACN / H2O / TFA = 90:9.0:1.0, v / v / v). Then 100 μΐ, of rinse solution was used to rinse 3 times, and the mixture was sonicated and shaken for 5 min (rinse solution: ACN / H2O / TFA = 90:9.9:0.1, v / v / v). This was to remove the non-phosphorylated peptides that were not bound to the material. Finally, 10 μΐ, of elution solution was added and shaken for 20 min to elute the phosphorylated peptides that were bound to the material (elution solution: 0.4 M NH4OH). The elution solution was collected and analyzed by MALDI-TOF to determine the enrichment of glycopeptides and phosphorylated peptides.

[0080] Figure 6 MSN-PEI-CS-PO3 2- -Ti 4+ glycopeptides and 19 phosphorylated peptides were enriched.

Claims

1. A method for preparing a mesoporous silica material modified with polyethyleneimine, chondroitin sulfate, characterized by: Comprising the following steps: a. Dissolve 1-2 g of chondroitin sulfate powder in 2-100 mL of ultrapure water, slowly add 10-20 g of methacrylic anhydride, then add a sodium hydroxide solution with a mass concentration of 1-40%, stir at room temperature for 1-24 hours, and stand in a 6°C refrigerator for 4-72 hours; b. Wash the product with water for 2-4 times, and freeze-dry to obtain chondroitin sulfate precursor CS-MA; c. Put 1-2 g of cetyltrimethylammonium chloride and 0.1-0.5 mL of triethanolamine in a reaction container in sequence, add 20-50 mL of deionized water, and reflux at 60-100°C for 1-24 hours, then add 1-5 mL of tetraethyl orthosilicate, and stir and reflux at 60-100°C for 1-24 hours: d. Wash the product with water for 2-4 times, and then wash with an organic reagent for 2-4 times; e. Vacuum dry at 60-100°C for 12-48 hours to obtain mesoporous silica MSN; f. Put 0.2-1 g of GLYMO silane reagent and 0.1-2 g of polyethyleneimine PEI in a reaction container in sequence, add 50-100 mL of N-N dimethylformamide, and stir and reflux at 70-100°C for 12-48 hours, then add mesoporous silica MSN, and stir and reflux at 70-100°C for 12-48 hours; g. Wash the product with N-N dimethylformamide for 2-4 times, and then wash with an organic reagent for 2-4 times; h. Wash with water for several times, and freeze-dry to obtain polyethyleneimine-modified mesoporous silica MSN-PEI; i. Further dissolve the polyethyleneimine-modified mesoporous silica MSN-PEI and the chondroitin sulfate precursor CS-MA in reagents respectively, and then mix, and react at room temperature under stirring for 24-48 hours; j. Wash the product with dimethyl sulfoxide for 2-4 times, wash with water for 2-4 times, and freeze-dry.

2. The method for preparing a mesoporous silica material modified with polyethyleneimine and chondroitin sulfate according to claim 1, characterized in that: The stirring in step a is magnetic stirring at a speed of 80-100 rpm.

3. The method for preparing a mesoporous silica material modified with polyethyleneimine and chondroitin sulfate according to claim 1, characterized in that: Before the water washing in step b, precipitate in ethanol for a period of time, then centrifuge and wash with water.

4. The method for preparing a mesoporous silica material modified with polyethyleneimine and chondroitin sulfate according to claim 1, characterized in that: The organic reagent in step d is selected from one or more of ethanol, methanol, acetonitrile, and acetone.

5. The method for preparing a mesoporous silica material modified with polyethyleneimine and chondroitin sulfate according to claim 1, characterized in that: The organic reagent in step g is selected from one or more of ethanol, methanol, acetonitrile, and acetone.

6. The method of claim 1, wherein the mesoporous silica material is modified with polyethyleneimine and chondroitin sulfate. The mass ratio of MSN-PEI to CS-MA in step i is 1-10:10-20.

7. The method for preparing a mesoporous silica material modified with polyethyleneimine and chondroitin sulfate according to claim 1, characterized in that: The reagent in step i is dimethyl sulfoxide or ultrapure water, and the stirring mode is magnetic stirring at a speed of 80-100 rpm.

8. A mesoporous silica material modified with polyethylenimine, chondroitin sulfate prepared according to the preparation method of claim 1, characterized by: The pore size of the mesoporous silica is 2-10 nm.

9. Use of the mesoporous silica material modified with polyethyleneimine and chondroitin sulfate in specific enrichment of glycopeptides in a hydrophilic mode.

10. Use according to claim 9, characterized in that: The glycopeptides are enriched in a column solid-phase extraction mode.

11. The method of claim 8, wherein the post-modification of the mesoporous silica material modified with polyethyleneimine and chondroitin sulfate is characterized by: Comprising the following steps: a. Add 50-100 mg of MSN-PEI-CS and 100-500 mg of N, N-carbonyldiimidazole dispersed in 20-100 mL of DMSO in sequence, stir at room temperature for 12-48 hours, then add 100-500 mg of aminomethyl phosphonic acid dispersed in 20-100 mL of DMF, and stir at room temperature for 1-12 hours; b. After the reaction is completed, it is washed thoroughly with organic reagent and freeze-dried to obtain MSN-PEI-CS-PO3 2- ; c. MSN-PEI-CS-PO3 2- Disperse in 100-200 mL Ti(SO4)2solution, stir the reaction at room temperature for 1-12 h, after the reaction is completed, wash with water, freeze-drying.

12. The method of claim 11, wherein the post-modification of the mesoporous silica material modified with polyethyleneimine, chondroitin sulfate is characterized by: The stirring in step a is magnetic stirring at a speed of 80-100 rpm.

13. The method of claim 11, wherein the post-modification is performed with polyethyleneimine, chondroitin sulfate modified mesoporous silica material. The organic reagent in step b is one or more of DMSO, DMF, ethanol, and methanol.

14. The method of claim 11, wherein the post-modification of the mesoporous silica material modified with polyethyleneimine and chondroitin sulfate is characterized by: The concentration of the Ti(SO4)2 solution in step c is 0.1-1 M; MSN-PEI-CS-PO3 2- The mass ratio to titanium sulfate is: 1-10: 100-200.

15. The use according to claim 9, characterized in that: The material is further functionalized to obtain a post-modified material for co-enrichment of glycopeptides and phosphorylated peptides.

16. The use according to claim 9, characterized in that: The material is surface-modified to form a post-modified material with hydrophilic groups and metal ion chelating groups, thereby combining hydrophilic interaction and metal ion affinity.

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