A captopril-modified hollow nanosilica sphere, its preparation method and application

Abstract

The present invention discloses a captopril-modified hollow nanosilica sphere and its preparation method and application, belonging to the technical field of nanomaterials. Among them, the preparation method of the captopril-modified hollow nanosilica sphere is as follows: using water as a solvent, F127 as a template agent, mesitylene as a micelle swelling agent, potassium sulfate as an auxiliary agent, and tetramethoxysilane and vinyltrimethoxysilane as silicon-based precursors, preparing vinyl-modified hollow nanosilica spheres through a sol-gel reaction; then, further modifying captopril on the hollow nanosilica spheres through a thiol-ene photoinitiated reaction. The beneficial effects of the present invention are as follows: The captopril-modified hollow nanosilica sphere is the first hydrophilic interaction chromatography stationary phase material prepared with captopril as a modifier monomer, having uniform particle size, good dispersibility, not easy to agglomerate, large specific surface area, and having good enrichment specificity and selectivity for N-glycopeptides, and can be used for the selective enrichment of N-glycopeptides in complex biological samples.

Description

Technical Field

[0001] The present invention relates to a hollow nanosilica sphere and its preparation method and application, specifically to a captopril-modified hollow nanosilica sphere and its preparation method and application, belonging to the technical field of nanomaterials. Background Art

[0002] Protein glycosylation is a common type of post-translational modification. More than 50% of proteins in the human body have glycosylation modification, which plays an important role in life activities such as immune response, signal transduction, and cell adhesion. Abnormal protein glycosylation modification is related to many diseases, including tumors, virus-induced infections, inflammations (such as rheumatoid arthritis), and allergic diseases. Clinical biomarkers and drug treatment targets for various diseases are also glycosylated proteins.

[0003] In mass spectrometry-based proteomics technology, protein identification mainly relies on peptide-level analysis. However, direct analysis of glycopeptides remains a challenge for two reasons:

[0004] (1) Glycosylation modification has macro and micro heterogeneity. The proportion of a single glycosylation modification type is low, and the proportion of glycosylated peptides after enzymatic digestion is even lower. The interference of non-glycosylated peptides is extremely large;

[0005] (2) Glycosylation modification reduces the ionization efficiency of peptides.

[0006] Therefore, the glycopeptide enrichment step before mass spectrometry analysis becomes an important link affecting the identification result. Enrichment strategies in glycoproteomics mainly include hydrophilic interaction chromatography, boronic acid affinity chromatography, lectin affinity chromatography, and hydrazide chemistry method, etc. Among them, hydrophilic interaction chromatography has become the most widely used method due to its advantages of no discrimination for different glycosylated peptides and no damage to the sugar chain part.

[0007] Captopril is an artificially synthesized antihypertensive drug belonging to angiotensin-converting enzyme inhibitors. It can perform molecular docking on angiotensin-converting enzyme in the human body through hydrogen bond interaction. Based on this background, the possibility of using captopril molecules as hydrophilic functionalization reagents is explored. The vinyl-functionalized hollow nanosilica spheres are modified with captopril as the functional monomer to prepare captopril-modified hollow nanosilica spheres, which are used as the stationary phase for hydrophilic interaction chromatography to investigate the application feasibility in the enrichment of N-glycosylated peptides. Summary of the Invention

[0008] The purpose of the present invention is to provide a captopril-modified hollow nanosilica sphere and its preparation method and application in the enrichment of N-glycopeptides in complex biological samples.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A preparation method of captopril-modified hollow nanosilica spheres. Without adding any acid-base reagents, using water as the solvent, F127 as the template agent, mesitylene as the micelle swelling agent, potassium sulfate as the auxiliary agent, and tetramethoxysilane and vinyltrimethoxysilane as the silicon precursors, vinyl-modified hollow nanosilica spheres are prepared through a sol-gel reaction; then, captopril is further modified on the hollow nanosilica spheres through a thiol-ene photoinitiated reaction to obtain captopril-modified hollow nanosilica spheres.

[0011] Preferably, the method for preparing vinyl-modified hollow nanosilica spheres through a sol-gel reaction is as follows: Add 1.75 g - 3.5 g of potassium sulfate, 0.5 g - 1.0 g of F127, and 0.58 mL - 1.16 mL of mesitylene into 30 mL - 60 mL of water, shake at 13.5 °C and 180 rpm for 12 h, then slowly dropwise add a mixture of 0.74 mL - 1.48 mL of tetramethoxysilane and 0.77 mL - 1.54 mL of vinyltrimethoxysilane while shaking, continue to shake at 13.5 °C and 180 rpm for 24 h, then transfer the reaction solution to a hydrothermal kettle, heat at 100 °C for 24 h, wash the obtained solid product with water and ethanol in sequence, and finally perform Soxhlet extraction with 200 mL - 400 mL of ethanol containing 2 mL - 4 mL of concentrated hydrochloric acid for 24 h, and dry under vacuum conditions to obtain vinyl-modified hollow nanosilica spheres.

[0012] Preferably, the method for further modifying captopril on the hollow nanosilica spheres through a thiol-ene photoinitiated reaction is as follows: Take 0.12 g of vinyl-modified hollow nanosilica spheres, ultrasonically disperse them in 20 mL of a mixed solution with a volume ratio of ethanol / water of 1 / 1, then add 0.2 g of captopril and 0.2 g of benzoin dimethyl ether, ultrasonically dissolve them, then carry out photoinitiation at a wavelength of 365 nm for 2 h, perform suction filtration, wash the product with water and ethanol in sequence, and dry under vacuum conditions to obtain captopril-modified hollow nanosilica spheres.

[0013] Captopril-modified hollow nanosilica spheres prepared by the aforementioned preparation method.

[0014] The application of the aforementioned captopril-modified hollow nanosilica spheres as a hydrophilic interaction chromatography stationary phase material for separating and enriching N-glycopeptides in biological samples; preferably, the biological samples are human or animal body fluid samples, tissue samples, or cell samples.

[0015] The beneficial effects of the present invention are as follows:

[0016] (1) The preparation method of captopril-modified hollow nanosilica spheres provided by the present invention:

[0017] (i) No acid-base reagents need to be added during the sol-gel reaction process;

[0018] (ii) The single micelle template method does not require additional methods such as deposition coating and sacrificial template to form a hollow structure;

[0019] (iii) The reaction conditions are mild, the requirements for reaction equipment are low, and it is suitable for large-scale preparation;

[0020] (iv) The functionalization step is simple and time-saving;

[0021] (2) The captopril-modified hollow nanosilica spheres prepared by the present invention:

[0022] (i) It is the first hydrophilic interaction chromatography stationary phase material prepared with captopril as the modification monomer;

[0023] (ii) It has uniform particle size, good dispersibility, is not easy to agglomerate, and has a large specific surface area;

[0024] (iii) It has good enrichment specificity and selectivity for N-glycopeptides, can be used as a stationary phase material for hydrophilic interaction chromatography, and is used for the selective enrichment of N-glycopeptides in complex biological samples. Compared with other materials (such as HTC-Glc-10%MPC), it has a comparable N-glycopeptide enrichment effect, but uses less amount and has a faster enrichment speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic flow chart for preparing captopril-modified hollow nanosilica spheres;

[0026] Figure 2 It is a transmission electron microscope image of the material. Among them, a is the transmission electron microscope image of SHS-V-1, b is the transmission electron microscope image of SHS-CAP-1, c is the transmission electron microscope image of SHS-V-2, and d is the transmission electron microscope image of SHS-CAP -2;

[0027] Figure 3 It is the nitrogen adsorption-desorption isotherm diagram and pore size distribution diagram of the material. Among them, a is the nitrogen adsorption-desorption isotherm diagram of SHS-V-1 and SHS-CAP-1, and b is the pore size distribution diagram of SHS-V-1 and SHS-CAP-1;

[0028] Figure 4 It is the infrared spectrum diagram and energy-dispersive X-ray spectrum diagram of the material. Among them, a is the infrared spectrum diagram of SHS, SHS-V-1 and SHS-CAP-1, and b is the energy-dispersive X-ray spectrum diagram of SHS-CAP-1;

[0029] Figure 5 It is the MALDI-TOF MS spectrum of the enzymatic hydrolysis product of immunoglobulin G. Among them, a is the MALDI-TOF MS spectrum before the enrichment of the enzymatic hydrolysis product, b is the MALDI-TOF MS spectrum after the enzymatic hydrolysis product is enriched by SHS-CAP-1, and c is the MALDI-TOF MS spectrum after the enzymatic hydrolysis product is enriched by SHS-CAP-1 and then the sugar chains are excised;

[0030] Figure 6 It is the result diagram of N-glycoproteome analysis after enriching N-glycopeptides in 2 μL of human serum sample with SHS-CAP-2. Among them, a is the analysis result diagram of N-glycosylated protein, b is the analysis result diagram of N-glycopeptide, and c is the analysis result diagram of specific N-glycosylation site;

[0031] Figure 7 It is the result diagram of N-glycoproteome analysis after enriching N-glycopeptides in 2 μL of human serum sample with HTC-Glc-10%MPC. Among them, a is the analysis result diagram of N-glycosylated protein, b is the analysis result diagram of N-glycopeptide, and c is the analysis result diagram of specific N-glycosylation site. Detailed implementation mode

[0032] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0033] I. Preparation of captopril-modified hollow nanosilica spheres

[0034] The present invention adopts a single micelle template method to prepare captopril-modified hollow nanosilica spheres. As Figure 1 shown, first, under the condition of not adding any acid-base reagents (neutral condition), using water as the solvent, using F127 (polyether ketone, molecular formula EO 106 PO 70 EO 106 ) as the template agent, using mesitylene (1,3,5-Trimethylbenzene, TMB) as the micelle swelling agent, using potassium sulfate as the auxiliary agent, using tetramethoxysilane (Tetramethoxysilane, TMOS) and vinyltrimethoxysilane (Vinyltrimethoxysilane, VTMS) as the silicon precursors, preparing vinyl-modified hollow nanosilica spheres through a sol-gel reaction; then, further modifying captopril on the hollow nanosilica spheres through a thiol-ene photoinitiated reaction to obtain captopril-modified hollow nanosilica spheres.

[0035] Example 1

[0036] 1.75 g of potassium sulfate, 0.5 g of F127 and 0.58 mL of mesitylene were added to 30 mL of water, and the mixture was shaken at 13.5 °C and 180 rpm for 12 h. Then, a mixture of 0.74 mL of tetramethoxysilane and 0.77 mL of vinyltrimethoxysilane was slowly added dropwise while shaking, and the mixture was continuously shaken at 13.5 °C and 180 rpm for 24 h. After that, the reaction solution was transferred to a hydrothermal reactor and heated at 100 °C for 24 h. The obtained solid product was washed successively with water and ethanol, and finally Soxhlet extracted with 200 mL of ethanol containing 2 mL of concentrated hydrochloric acid (concentration: 12 mol / L) for 24 h, and dried under vacuum to obtain vinyl-modified hollow nanosilica spheres, denoted as SHS-V-1.

[0037] 0.12 g of the previously prepared SHS-V-1 was ultrasonically dispersed in 20 mL of a mixed solution of ethanol / water with a volume ratio of 1 / 1. Then, 0.2 g of captopril and 0.2 g of benzoin dimethyl ether (initiator) were added, and the mixture was ultrasonically dissolved. After that, photoinitiation was carried out at a wavelength of 365 nm for 2 h. The product was filtered by suction, and the product was successively rinsed with water and ethanol and dried under vacuum to obtain captopril-modified hollow nanosilica spheres, denoted as SHS-CAP-1.

[0038] Example 2

[0039] 3.5 g of potassium sulfate, 1.0 g of F127 and 1.16 mL of mesitylene were added to 60 mL of water, and the mixture was shaken at 13.5 °C and 180 rpm for 12 h. Then, a mixture of 1.48 mL of tetramethoxysilane and 1.54 mL of vinyltrimethoxysilane was slowly added dropwise while shaking, and the mixture was continuously shaken at 13.5 °C and 180 rpm for 24 h. After that, the reaction solution was transferred to a hydrothermal reactor and heated at 100 °C for 24 h. The obtained solid product was washed successively with water and ethanol, and finally Soxhlet extracted with 400 mL of ethanol containing 4 mL of concentrated hydrochloric acid (concentration: 12 mol / L) for 24 h, and dried under vacuum to obtain vinyl-modified hollow nanosilica spheres, denoted as SHS-V-2.

[0040] 0.12 g of the previously prepared SHS-V-2 was ultrasonically dispersed in 20 mL of a mixed solution of ethanol / water with a volume ratio of 1 / 1. Then, 0.2 g of captopril and 0.2 g of benzoin dimethyl ether (initiator) were added, and the mixture was ultrasonically dissolved. After that, photoinitiation was carried out at a wavelength of 365 nm for 2 h. The product was filtered by suction, and the product was successively rinsed with water and ethanol and dried under vacuum to obtain captopril-modified hollow nanosilica spheres, denoted as SHS-CAP-2.

[0041] Comparative Example

[0042] To compare the changes in functional groups and elements during the material preparation process, a comparative example without adding vinyltrimethoxysilane was used to prepare hollow nanosilica spheres without vinyl modification. Specifically:

[0043] Take 1.75 g of potassium sulfate, 0.5 g of F127, and 0.58 mL of mesitylene and add them to 30 mL of water. Shake at 13.5 °C and 180 rpm for 12 h, then slowly add 1.48 mL of tetramethoxysilane dropwise while shaking, and continue to shake at 13.5 °C and 180 rpm for 24 h. Then transfer the reaction solution to a hydrothermal autoclave and heat at 100 °C for 24 h. Wash the obtained solid product with water and ethanol in sequence, and finally perform Soxhlet extraction with 200 mL of ethanol containing 2 mL of concentrated hydrochloric acid (concentration 12 mol / L) for 24 h, and dry under vacuum to obtain hollow nanosilica spheres without vinyl modification, denoted as SHS.

[0044] II. Product Characterization

[0045] 1. Characterize the morphological structure of the material using a transmission electron microscope

[0046] Use a transmission electron microscope to characterize the morphological structures of SHS-V-1 and SHS-CAP-1 prepared in Example 1 and SHS-V-2 and SHS-CAP-2 prepared in Example 2.

[0047] The characterization results of the morphological structure are shown in Figure 2 . From Figure 2 it can be seen that:

[0048] (1) Both SHS-V-1 (a) prepared in Example 1 and SHS-V-2 (c) prepared in Example 2 have typical hollow nanostructures;

[0049] (2) After being modified with captopril, SHS-CAP-1 (b) shows the same hollow spherical structure as SHS-V-1 (a), and SHS-CAP-2 (d) shows the same hollow spherical structure as SHS-V-2 (c), that is, the morphological structure of the material does not change significantly after being modified with captopril;

[0050] (3) The particle sizes of SHS-CAP-1 (b) prepared in Example 1 and SHS-CAP-2 (d) prepared in Example 2 are about 17 nm, the cavity diameters are about 10 nm, and the shell thicknesses are about 3.5 nm.

[0051] 2. Characterize the properties such as the pore structure of the material through physical adsorption experiments

[0052] The pore structure, pore size distribution, specific surface area, total pore volume, etc. of SHS-V-1 and SHS-CAP-1 prepared in Example 1 were characterized by physical adsorption experiments.

[0053] The measurement results of the nitrogen adsorption-desorption isotherm are shown in Figure 3 Figure (a). From Figure 3 Figure (a), it can be seen that:

[0054] (1) According to the IUPAC classification, SHS-V-1 simultaneously exhibits the characteristics of both Type I and Type IV isotherms: at lower relative pressures, the sharp increase in nitrogen adsorption reflects the presence of micropores on the hollow nanosilica shell, while the hysteresis loop at higher relative pressures is related to the presence of mesopores. The two hysteresis loops in the isotherm come from the hollow internal cavity structure and the packing of nanosilica spheres, respectively;

[0055] (2) Compared with SHS-V-1, SHS-CAP-1 does not show a sharp increase in nitrogen adsorption in the lower relative pressure range, and the first hysteresis loop disappears at higher relative pressures, indicating that the micropores on the shell and the mesopore structure of the hollow cavity are not measured.

[0056] The measurement results of the pore size distribution are shown in Figure 3 Figure (b). From Figure 3 Figure (b), it can be seen that:

[0057] (1) SHS-V-1 simultaneously has three different pore structures with different pore sizes (consistent with the results of the nitrogen adsorption-desorption isotherm);

[0058] (2) For SHS-CAP-1, the first peak corresponding to the micropores on the shell disappears, and the second peak corresponding to the hollow cavity also becomes less obvious. This may be because the reaction between captopril and the vinyl groups on the surface of the hollow silica sphere during the modification process blocks the pore structure on the shell, thereby hindering the nitrogen adsorption of the mesopore structure corresponding to the hollow cavity (judging from the results of transmission electron microscopy, SHS-CAP-1 still has a hollow structure).

[0059] The Brunauer-Emmett-Teller model was used to calculate the specific surface areas of SHS-V-1 and SHS-CAP-1. The results are as follows: the specific surface area of SHS-V-1 is 543 m 2 / g, and the specific surface area of SHS-CAP-1 is 190 m 2 / g.

[0060] The total pore volumes of SHS-V-1 and SHS-CAP-1 were calculated using the nitrogen adsorption amount at a relative pressure of 0.99. The results are as follows: the total pore volume of SHS-V-1 is 1.76 cm 3 / g, and the total pore volume of SHS-CAP-1 is 1.32 cm3 / g.

[0061] The pore size of SHS-V-1 was calculated according to the density functional theory (DFT). Results: The micropore diameter on the shell of SHS-V-1 was about 2.1 nm, and the diameter of the hollow cavity was about 8.2 nm.

[0062] 3. Verify the changes in functional groups and elements during the material preparation process by Fourier transform-infrared spectroscopy and energy-dispersive X-ray spectroscopy

[0063] The Fourier transform-infrared spectroscopy detection results of SHS, SHS-V-1, and SHS-CAP-1 are shown in Figure 4 (a). From Figure 4 (a), it can be seen that:

[0064] (1) By comparing the infrared spectra of SHS and SHS-V-1, it can be found that new absorption peaks at 3067 cm -1 , 3028 cm -1 , 2963 cm -1 , 1604 cm -1 , 1412 cm -1 and 969 cm -1 appear in the infrared spectrum of SHS-V-1, which are attributed to the asymmetric stretching vibration of =CH2 in vinyl, the stretching vibration of =CH-, the symmetric stretching vibration of =CH2, the stretching vibration of C=C, the bending vibration of =CH2, and the bending vibration of =CH-, respectively, indicating that vinyl-modified hollow nanosilica spheres SHS-V-1 were successfully prepared through the co-hydrolysis and polycondensation process of tetramethoxysilane and vinyltrimethoxysilane;

[0065] (2) By comparing the infrared spectra of SHS-V-1 and SHS-CAP-1, it can be found that the six absorption peaks corresponding to vinyl in the infrared spectrum of SHS-V-1 do not appear in the infrared spectrum of SHS-CAP-1, indicating that vinyl has been completely reacted;

[0066] (3) New absorption peaks at 2980 cm -1 , 1739 cm -1 and 1625 cm -1 appear in the infrared spectrum of SHS-CAP-1, which are attributed to the stretching vibration of C-H in methyl, the stretching vibration of C=O in carboxyl, and the stretching vibration of C=O in tertiary amide, respectively, indicating that captopril has successfully reacted with vinyl in SHS-V-1 and has been modified onto SHS-V-1.

[0067] The energy-dispersive X-ray spectroscopy detection results of SHS-CAP-1 are shown in Figure 4 (b). From Figure 4It can be seen from Fig. (b) that peaks corresponding to carbon, oxygen, silicon, nitrogen, and sulfur elements appear in the spectrum, further indicating the successful modification of captopril.

[0068] III. Application of the product as a hydrophilic interaction chromatography stationary phase material

[0069] 1. Preparation of protease-digested sample

[0070] Dissolve 2 mg of immunoglobulin G in 1 mL of ammonium bicarbonate solution containing 8 mol / L urea, pH = 8.2, and a concentration of 100 mmol / L. Then add 80 μmol of DL-1,4-dithiothreitol and react at a constant temperature in a 37 °C water bath for 2 h. After that, add 40 μmol of 2-iodoacetamide and react in the dark for 40 min. Next, add 7 mL of ammonium bicarbonate solution with pH = 8.2 and a concentration of 100 mmol / L and 80 μg of trypsin, and digest in a 37 °C water bath for 18 h. Finally, desalt to obtain the immunoglobulin G protease-digested sample, aliquot and freeze-dry, and store at -20 °C for later use.

[0071] 2. Enrich N-glycopeptides in the immunoglobulin G protease-digested sample using SHS-CAP-1 prepared in Example 1

[0072] Weigh 2 mg of SHS-CAP-1 prepared in Example 1 into a centrifuge tube, equilibrate with 200 μL of loading solution (acetonitrile, water, trifluoroacetic acid mixed in a volume ratio of 85:14:1), then add 200 μL of loading solution containing 9 μg of immunoglobulin G protease-digested sample, shake at 25 °C and 1500 rpm for 30 min, then centrifuge and discard the supernatant. Next, wash SHS-CAP-1 three times with 200 μL of loading solution (purpose: remove non-specifically adsorbed peptide segments), 5 min each time. Subsequently, elute the N-glycopeptides adsorbed on SHS-CAP-1 with 200 μL of elution solution (acetonitrile, water, trifluoroacetic acid mixed in a volume ratio of 30:69:1) for 20 min, centrifuge. One part of the eluate is directly analyzed by matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS), and the other part of the eluate is freeze-dried, deglycosylated with PNGase F enzyme by conventional techniques in the art, and then further analyzed by MALDI-TOF MS.

[0073] Among them, the method for mass spectrometry analysis using MALDI-TOF MS is as follows:

[0074] Drop 0.5 μL of the sample onto the MALDI target. After natural drying, cover the sample spot with 0.5 μL of a 2,5-dihydroxybenzoic acid solution with a concentration of 25 mg / mL as the matrix. After complete natural drying, send it to MALDI-TOF MS for mass spectrometry analysis.

[0075] The MALDI-TOF MS spectra of the immunoglobulin G enzymatic digestion sample before enrichment, the MALDI-TOF MS spectra after enrichment with SHS-CAP-1, and the MALDI-TOF MS spectra after excision of the sugar chains after enrichment are shown in Figure 5 . From Figure 5 it can be seen that:

[0076] (1) Before enrichment (a), the signals of a large number of high-abundance non-glycosylated peptides (mass-to-charge ratio below 2200) occupy most of the spectrum, and the signals of N-glycopeptides (mass-to-charge ratio above 2200) are hardly detectable;

[0077] (2) After enriching the immunoglobulin G enzymatic digestion sample with SHS-CAP-1 under the loading solution conditions of acetonitrile, water, and trifluoroacetic acid (85 / 14 / 1, v / v / v) (b), the complexity of the sample is greatly reduced, the signals of non-glycosylated peptides are almost completely removed, and the signal intensity of N-glycopeptides is greatly increased. After mass-to-charge ratio comparison, 24 N-glycopeptides can be detected from 1.5 pmol of the immunoglobulin G enzymatic digestion sample after enrichment with SHS-CAP-1 (Table 1), indicating that SHS-CAP-1 shows high specificity in N-glycopeptide enrichment;

[0078] (3) After digesting the sugar chains of the enriched peptides with PNGase F (c), only two peaks appear in the MALDI-TOF MS spectrum, with mass-to-charge ratios of 1158 and 1190 respectively, corresponding to two deglycosylated modified peptides in the immunoglobulin G enzymatic digestion sample, further confirming the high specificity of SHS-CAP-1 in N-glycopeptide enrichment.

[0079] Table 1 Details of N-glycopeptides enriched from 1.5 pmol of human immunoglobulin G enzymatic digestion sample using SHS-CAP-1

[0080]

[0081] 3. Enrich N-glycopeptides in human serum with SHS-CAP-2 prepared in Example 2

[0082] To investigate the feasibility of using captopril-modified hollow nanosilica spheres for the enrichment of N-glycopeptides in actual biological samples (e.g., human body fluids, tissues and cells; animal body fluids, tissues and cells), the N-glycopeptides in 2 μL of human serum were enriched using SHS-CAP-2 prepared in Example 2, and N-glycoproteomic analysis was performed on the LC-MS / MS results.

[0083] The enrichment process of N-glycopeptides in the serum sample was similar to that in the immunoglobulin G digestion sample mentioned above. The differences were as follows: the amount of SHS-CAP-2 used was 5 mg, the amount of serum used was 2 μL, the volume of the loading solution was 400 μL, and the enriched N-glycopeptides were lyophilized and the sugar chains were removed before LC-MS / MS analysis.

[0084] After the N-glycopeptides in 2 μL of human serum sample were enriched three times in duplicate using SHS-CAP-2, the results of N-glycoproteomic analysis are shown in Figure 6 . As can be seen from Figure 6 : After enrichment of the human serum sample with SHS-CAP-2, 314 specific N-glycosylation peptides and 165 specific N-glycosylation sites from 84 N-glycoproteins were identified by LC-MS / MS. The detailed information of the 84 N-glycoproteins and 314 specific N-glycosylation peptides is shown in Table 2.

[0085] Table 2 Detailed information of N-glycoproteins and specific N-glycosylation peptides enriched from 2 μL of human serum sample using SHS-CAP-2.

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] 4. Enrichment of N-glycopeptides in human serum samples using the published HTC-Glc-10% MPC

[0098] For comparison, the published HTC-Glc-10% MPC (Li et al., One-step preparation of zwitterionic-rich hydrophilic hydrothermal carbonaceous materials for enrichment of N-glycopeptides, ACS Sustainable Chemistry & Engineering, 2019, 7(13): 11511-11520) was used to enrich N-glycopeptides in 2 μL of human serum samples, and N-glycoproteomic analysis was performed on the LC-MS / MS results.

[0099] The process of enriching N-glycopeptides in human serum samples using HTC-Glc-10% MPC is different from the process of enriching N-glycopeptides in human serum samples using SHS-CAP-2 as follows: the amount of HTC-Glc-10% MPC used is 10 mg, the loading time is 1 h, the washing time is 10 min each time, and the elution time is 40 min.

[0100] After three repeated enrichments of N-glycopeptides in 2 μL of human serum samples using HTC-Glc-10% MPC, the results of N-glycoproteomic analysis are shown in Figure 7 . It can be seen from Figure 7 that: after enrichment of human serum samples with HTC-Glc-10% MPC, 285 specific N-glycopeptides from 92 N-glycoproteins and 175 specific N-glycosylation sites were identified by LC-MS / MS.

[0101] Comparison Figure 6 and Figure 7 shows that: the SHS-CAP-2 prepared in the present invention has a comparable N-glycopeptide enrichment effect to the published HTC-Glc-10% MPC, but the former has a smaller amount used and a faster enrichment speed.

[0102] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not limitations on the implementation modes of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation modes here. Any obvious changes or modifications derived from the technical solution of the present invention still fall within the protection scope of the present invention.

Claims

1. Application of captopril-modified hollow nanosilica spheres as a hydrophilic interaction chromatography stationary phase material, characterized in that, For separating and enriching N-glycopeptides in a biological sample; the preparation method of the captopril-modified hollow nanosilica spheres is as follows: without adding any acid-base reagents, using water as the solvent, F127 as the template agent, mesitylene as the micelle swelling agent, potassium sulfate as the auxiliary agent, and tetramethoxysilane and vinyltrimethoxysilane as the silicon precursors, vinyl-modified hollow nanosilica spheres are prepared by a sol-gel reaction; then, captopril is further modified on the hollow nanosilica spheres through a thiol-ene photoinitiated reaction. Take 0.12 g of the vinyl-modified hollow nanosilica spheres and ultrasonically disperse them in 20 mL of a mixed solution with a volume ratio of ethanol / water of 1 / 1, then add 0.2 g of captopril and 0.2 g of benzoin dimethyl ether, ultrasonically dissolve them, and then carry out photoinitiation at a wavelength of 365 nm for 2 h, filter by suction, wash the product successively with water and ethanol, and dry it under vacuum conditions to obtain the captopril-modified hollow nanosilica spheres.

2. The application according to claim 1, characterized in that, The method for preparing vinyl-modified hollow nanosilica spheres by a sol-gel reaction is specifically as follows: Take 1.75 g - 3.5 g of potassium sulfate, 0.5 g - 1.0 g of F127, and 0.58 mL - 1.16 mL of mesitylene and add them to 30 mL - 60 mL of water, shake at 13.5 °C and 180 rpm for 12 h, then slowly add dropwise a mixture of 0.74 mL - 1.48 mL of tetramethoxysilane and 0.77 mL - 1.54 mL of vinyltrimethoxysilane while shaking, continue to shake at 13.5 °C and 180 rpm for 24 h, then transfer the reaction solution to a hydrothermal kettle and heat at 100 °C for 24 h, wash the obtained solid product successively with water and ethanol, and finally perform Soxhlet extraction with 200 mL - 400 mL of ethanol containing 2 mL - 4 mL of concentrated hydrochloric acid for 24 h, and dry it under vacuum conditions to obtain the vinyl-modified hollow nanosilica spheres.

3. The application according to claim 1, characterized in that, The biological sample is a body fluid sample, tissue sample or cell sample of a human or an animal.

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