Captopril-modified hollow nano-silicon sphere as well as preparation method and application of captopril-modified hollow nano-silicon sphere

Abstract

The invention discloses captopril-modified hollow nano silicon spheres as well as a preparation method and application thereof, and belongs to the technical field of nano materials. The preparation method of the captopril-modified hollow nanometer silicon spheres comprises the following steps: by taking water as a solvent, F127 as a template agent, mesitylene as a micelle expanding agent, potassium sulfate as an auxiliary agent and tetramethoxysilane and vinyl trimethoxysilane as silicon-based precursors, preparing vinyl-modified hollow nanometer silicon spheres through a sol-gel reaction; then, captopril is further modified on the hollow nano silicon spheres through a sulfydryl-alkene photo-initiation reaction. The captopril-modified hollow nanometer silicon sphere has the beneficial effects that the captopril-modified hollow nanometer silicon sphere is the first hydrophilic interaction chromatography stationary phase material prepared by taking captopril as a modification monomer, is uniform in particle size, good in dispersity, not easy to agglomerate and large in specific surface area, has very good enrichment specificity and selectivity on N-glycopeptide, and can be used for preparing the hydrophilic interaction chromatography stationary phase material. The method can be used for selective enrichment of N-glycopeptide in complex biological samples.

Description

Technical Field

[0001] The invention relates to a hollow nano silicon sphere and a preparation method and application thereof, in particular to a captopril-modified hollow nano silicon sphere and a preparation method and application thereof, belonging to the technical field of nano materials. Background Art

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

[0003] In mass spectrometry-based proteomics technology, protein identification currently relies mainly on peptide-level analysis. However, direct analysis of glycopeptides remains a challenge for two reasons: (1) Glycosylation modification has macroscopic and microscopic heterogeneity. The proportion of single glycosylation modification type is low, and the proportion of glycosylated peptides after enzymatic hydrolysis is even lower. The interference of non-glycosylated peptides is great; (2) Glycosylation modification reduces the ionization efficiency of peptide fragments.

[0004] Therefore, the glycopeptide enrichment step before mass spectrometry analysis becomes an important link that affects the identification results. The main enrichment strategies in glycosylation proteomics include hydrophilic interaction chromatography, boric acid affinity chromatography, lectin affinity chromatography, and hydrazide chemistry. Among them, hydrophilic interaction chromatography has become the most widely used method due to its advantages such as non-discrimination for different glycosylated peptides and no damage to the sugar chain part.

[0005] Captopril is a synthetic antihypertensive drug and an angiotensin converting enzyme inhibitor. It can dock the angiotensin converting enzyme in the human body through hydrogen bond interactions. Based on this background, the possibility of captopril molecule as a hydrophilic functionalization reagent was explored. Vinyl-functionalized hollow nano-silica spheres were modified with captopril as the functional monomer to prepare captopril-modified hollow nano-silica spheres, which were used as the stationary phase of hydrophilic interaction chromatography to investigate its feasibility in the enrichment of N-glycosylated peptides. Summary of the invention

[0006] The purpose of the present invention is to provide a captopril-modified hollow nano-silica sphere and a preparation method thereof and an application thereof in enriching N-glycopeptides in complex biological samples.

[0007] In order to achieve the above object, the present invention adopts the following technical solution: The invention discloses a method for preparing hollow nano-silicon spheres modified with captopril. The method comprises the following steps: 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 to prepare hollow nano-silicon spheres modified with vinyl through a sol-gel reaction without adding any acid or alkali reagents; and then further modifying the hollow nano-silicon spheres with captopril through a thiol-ene photoinitiator reaction to obtain hollow nano-silicon spheres modified with captopril.

[0008] Preferably, the method for preparing vinyl-modified hollow nano-silica spheres by sol-gel reaction is as follows: 1.75g-3.5g potassium sulfate, 0.5g-1.0g F127 and 0.58mL-1.16mL mesitylene are added to 30mL-60mL water, shaken at 13.5°C and 180rpm for 12h, and then a mixture of 0.74mL-1.48mL tetramethoxysilane and 0.77mL-1.54mL vinyltrimethoxysilane is slowly added dropwise while shaking, and the mixture is continued to be shaken at 13.5°C and 180rpm for 24h, and then the reaction solution is transferred to a hydrothermal autoclave, heated at 100°C for 24h, and the obtained solid product is washed with water and ethanol in sequence, and finally Soxhlet extraction is performed with 200mL-400mL ethanol containing 2mL-4mL concentrated hydrochloric acid for 24h, and dried under vacuum to obtain vinyl-modified hollow nano-silica spheres.

[0009] Preferably, the method for further modifying captopril on the hollow nano-silica spheres by thiol-ene photoinitiator reaction is as follows: 0.12 g of vinyl-modified hollow nano-silica spheres are ultrasonically dispersed in 20 mL of a mixed solution of ethanol / water with a volume ratio of 1 / 1, and then 0.2 g of captopril and 0.2 g of benzoin dimethyl ether are added, ultrasonically dissolved, and then photoinitiated at a wavelength of 365 nm for 2 h, filtered, and the product is successively rinsed with water and ethanol, and dried under vacuum conditions to obtain captopril-modified hollow nano-silica spheres.

[0010] The captopril-modified hollow nano-silicon spheres are prepared by the above-mentioned preparation method.

[0011] The aforementioned captopril-modified hollow nano-silica spheres are used as hydrophilic interaction chromatography stationary phase materials to separate and enrich N-glycopeptides in biological samples; preferably, the biological samples are human or animal body fluid samples, tissue samples or cell samples.

[0012] The present invention is beneficial in that: (1) The preparation method of the captopril-modified hollow nano-silicon spheres provided by the present invention: (i) No acid or base reagents need to be added during the sol-gel reaction; (ii) The single micelle template method does not require additional deposition coating and sacrificial template methods to form hollow structures; (iii) The reaction conditions are mild, the requirements for reaction equipment are low, and it is suitable for large-scale preparation; (iv) The functionalization step is simple and time-saving; (2) Captopril-modified hollow nano-silicon spheres prepared by the present invention: (i) It is the first hydrophilic interaction chromatography stationary phase material prepared with captopril as the modified monomer; (ii) Uniform particle size, good dispersibility, not easy to agglomerate, and large specific surface area; (iii) It has good enrichment specificity and selectivity for N-glycopeptides and can be used as a stationary phase material for hydrophilic interaction chromatography 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 requires less usage and a faster enrichment speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the process of preparing captopril-modified hollow nano-silicon spheres; Figure 2 are transmission electron micrographs of the materials, wherein a is a transmission electron micrograph of SHS-V-1, b is a transmission electron micrograph of SHS-CAP-1, c is a transmission electron micrograph of SHS-V-2, and d is a transmission electron micrograph of SHS-CAP -2; Figure 3 It is the nitrogen adsorption-desorption isotherm diagram and pore size distribution diagram of the material, where 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; Figure 4 are the infrared spectra and energy dispersive X-ray spectra of the materials, where a is the infrared spectra of SHS, SHS-V-1 and SHS-CAP-1, and b is the energy dispersive X-ray spectra of SHS-CAP-1; Figure 5 : are MALDI-TOF MS spectra of the enzymatic hydrolysis products of immunoglobulin G, wherein a is the MALDI-TOF MS spectrum of the enzymatic hydrolysis products before enrichment, b is the MALDI-TOF MS spectrum of the enzymatic hydrolysis products after enrichment by SHS-CAP-1, and c is the MALDI-TOF MS spectrum of the enzymatic hydrolysis products after enrichment by SHS-CAP-1 and then removal of sugar chains; Figure 6The following is a graph showing the analysis results of N-glycosylated proteome after enriching N-glycopeptides in 2 μL human serum samples using SHS-CAP-2, where a is the analysis result graph of N-glycosylated proteins, b is the analysis result graph of N-glycosylated peptide segments, and c is the analysis result graph of specific N-glycosylation sites; Figure 7 The figure is the analysis result of N-glycosylation proteome after enriching N-glycopeptides in 2 μL human serum sample with HTC-Glc-10% MPC, where a is the analysis result of N-glycosylated protein, b is the analysis result of N-glycosylated peptide segment, and c is the analysis result of specific N-glycosylation site. DETAILED DESCRIPTION

[0014] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0015] 1. Preparation of Captopril-modified Hollow Nanosilica Spheres The present invention adopts a single micelle template method to prepare captopril-modified hollow nano-silicon spheres. Figure 1 As shown, first, without adding any acid or alkali reagent (under neutral conditions), water was used as solvent and F127 (polyether ketone, molecular formula is EO 106 PO 70 EO 106 ) was used as a template, 1,3,5-Trimethylbenzene (TMB) was used as a micelle swelling agent, potassium sulfate was used as an auxiliary agent, tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) were used as silicon-based precursors, and vinyl-modified hollow nano-silica spheres were prepared by a sol-gel reaction; then, captopril was further modified on the hollow nano-silica spheres by a thiol-ene photoinitiator reaction to obtain captopril-modified hollow nano-silica spheres.

[0016] Example 1

[0017] 1.75 g potassium sulfate, 0.5 g F127 and 0.58 mL mesitylene were added to 30 mL water, and the mixture was shaken at 13.5 ° C and 180 rpm for 12 h. Then, a mixture of 0.74 mL tetramethoxysilane and 0.77 mL vinyltrimethoxysilane was slowly added dropwise while shaking, and the mixture was continued to be shaken at 13.5 ° C and 180 rpm for 24 h. After that, the reaction solution was transferred to a hydrothermal autoclave and heated at 100 ° C for 24 h. The obtained solid product was washed with water and ethanol in turn, and finally Soxhlet extracted with 200 mL ethanol containing 2 mL concentrated hydrochloric acid (concentration of 12 mol / L) for 24 h, and dried under vacuum to obtain vinyl-modified hollow nanosilica spheres, which were recorded as SHS-V-1.

[0018] 0.12 g of the previously prepared SHS-V-1 was ultrasonically dispersed in 20 mL of a mixed solution of ethanol / water in a ratio of 1 / 1 (volume ratio), and then 0.2 g of captopril and 0.2 g of benzoin dimethyl ether (initiator) were added and dissolved ultrasonically. The mixture was then photo-initiated at a wavelength of 365 nm for 2 h and filtered. The product was sequentially washed with water and ethanol and dried under vacuum to obtain captopril-modified hollow nanosilica spheres, which were designated as SHS-CAP-1.

[0019] Example 2

[0020] 3.5 g potassium sulfate, 1.0 g F127 and 1.16 mL mesitylene were added to 60 mL water, and the mixture was shaken at 13.5 ° C and 180 rpm for 12 h. Then, a mixture of 1.48 mL tetramethoxysilane and 1.54 mL vinyltrimethoxysilane was slowly added dropwise while shaking, and the mixture was continued to be shaken at 13.5 ° C and 180 rpm for 24 h. After that, the reaction solution was transferred to a hydrothermal autoclave and heated at 100 ° C for 24 h. The solid product was washed with water and ethanol in turn, and finally Soxhlet extracted with 400 mL ethanol containing 4 mL concentrated hydrochloric acid (concentration of 12 mol / L) for 24 h, and dried under vacuum to obtain vinyl-modified hollow nanosilica spheres, which were recorded as SHS-V-2.

[0021] 0.12 g of the previously prepared SHS-V-2 was ultrasonically dispersed in 20 mL of a mixed solution of ethanol / water in a ratio of 1 / 1 (volume ratio), and then 0.2 g of captopril and 0.2 g of benzoin dimethyl ether (initiator) were added and dissolved ultrasonically. The mixture was then photo-initiated at a wavelength of 365 nm for 2 h and filtered. The product was sequentially washed with water and ethanol and dried under vacuum to obtain captopril-modified hollow nanosilica spheres, which were designated as SHS-CAP-2.

[0022] Comparative Example In order to compare the functional group changes and element changes during the material preparation process, the hollow nano-silicon spheres without vinyl modification were prepared by taking no addition of vinyl trimethoxysilane as a comparative example. Specifically: 1.75 g potassium sulfate, 0.5 g F127 and 0.58 mL mesitylene were added to 30 mL water, and the mixture was shaken at 13.5 ° C and 180 rpm for 12 h. Then, 1.48 mL tetramethoxysilane was slowly added dropwise while shaking, and the mixture was continued to be shaken at 13.5 ° C and 180 rpm for 24 h. After that, the reaction solution was transferred to a hydrothermal autoclave and heated at 100 ° C for 24 h. The obtained solid product was washed with water and ethanol in turn, and finally Soxhlet extracted with 200 mL ethanol containing 2 mL concentrated hydrochloric acid (concentration of 12 mol / L) for 24 h, and dried under vacuum to obtain hollow nanosilica spheres without vinyl modification, which were recorded as SHS.

[0023] 2. Product Characterization 1. Characterize the morphology and structure of the material using transmission electron microscopy The morphology and structure of SHS-V-1 and SHS-CAP-1 prepared in Example 1 and SHS-V-2 and SHS-CAP-2 prepared in Example 2 were characterized by transmission electron microscopy.

[0024] The characterization results of the morphology and structure are shown in Figure 2 .Depend on Figure 2 It can be seen that: (1) SHS-V-1 (a) prepared in Example 1 and SHS-V-2 (c) prepared in Example 2 both have typical hollow nanostructures; (2) After modification with captopril, SHS-CAP-1 (b) exhibited the same hollow spherical structure as SHS-V-1 (a), and SHS-CAP-2 (d) exhibited the same hollow spherical structure as SHS-V-2 (c), indicating that the morphology and structure of the materials did not change significantly after modification with captopril; (3) The particle size of SHS-CAP-1 (b) prepared in Example 1 and SHS-CAP-2 (d) prepared in Example 2 was about 17 nm, the cavity diameter was about 10 nm, and the shell thickness was about 3.5 nm.

[0025] 2. Characterize the pore structure and other properties of the material through physical adsorption experiments 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.

[0026] The results of nitrogen adsorption-desorption isotherm measurements are shown in Figure 3 (a). Figure 3From (a), we can see that: (1) According to the IUPAC classification, SHS-V-1 has the characteristics of both type I and type IV isotherms: the steep increase in nitrogen adsorption at lower relative pressure reflects the presence of micropores on the shell of the hollow nanosilicon spheres, while the hysteresis loop at higher relative pressure is related to the presence of mesopores. The two hysteresis loops in the isotherm come from the hollow internal cavity structure and the accumulation of nanosilicon spheres, respectively. (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 mesoporous structure of the hollow cavity are not measured.

[0027] The pore size distribution measurement results are shown in Figure 3 (b). Figure 3 From (b), we can see that: (1) SHS-V-1 has three pore structures with different pore sizes (consistent with the results of the nitrogen adsorption-desorption isotherm curve); (2) The first peak corresponding to the micropores on the shell of SHS-CAP-1 disappeared, and the second peak corresponding to the hollow cavity also became less obvious. This may be because the reaction between captopril and the vinyl group on the surface of the hollow silica sphere during the modification process blocked the pore structure on the shell, thereby hindering the adsorption of nitrogen by the mesoporous structure corresponding to the hollow cavity (combined with the results of transmission electron microscopy, SHS-CAP-1 still has a hollow structure).

[0028] The specific surface area of ​​SHS-V-1 and SHS-CAP-1 was calculated using the Brunauer-Emmett-Teller model. The results showed that the specific surface area of ​​SHS-V-1 was 543 m 2 / g, the specific surface area of ​​SHS-CAP-1 is 190m 2 / g.

[0029] The total pore volume of SHS-V-1 and SHS-CAP-1 was calculated using the nitrogen adsorption at a relative pressure of 0.99. The results showed that the total pore volume of SHS-V-1 was 1.76 cm 3 / g, and the total pore volume of SHS-CAP-1 is 1.32 cm 3 / g.

[0030] The pore size of SHS-V-1 was calculated based on density functional theory (DFT). The results showed that the diameter of the micropores on the shell of SHS-V-1 was about 2.1 nm, and the diameter of the hollow cavity was about 8.2 nm.

[0031] 3. Verify the functional group changes and element changes during material preparation by Fourier transform-infrared spectroscopy and energy dispersive X-ray spectroscopy The Fourier transform-infrared spectroscopy test results of SHS, SHS-V-1 and SHS-CAP-1 are shown in Figure 4 (a). Figure 4 From (a), we can see that: (1) By comparing the infrared spectra of SHS and SHS-V-1, it can be found that a new infrared spectroscopy at 3067 cm -1 、3028cm -1 、2963cm -1 、1604cm -1 、1412cm -1 and 969cm -1 The absorption peaks are attributed to =CH 2 Asymmetric stretching vibration, =CH- stretching vibration, =CH 2 Symmetric stretching vibration, C=C stretching vibration, =CH 2 The bending vibration of =CH- indicates that the vinyl-modified hollow nanosilica spheres SHS-V-1 were successfully prepared through the co-hydrolysis and polycondensation process of tetramethoxysilane and vinyltrimethoxysilane; (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 the vinyl has been completely reacted; (3) A new region at 2980 cm-1 was added to the infrared spectrum of SHS-CAP-1. -1 、1739cm -1 and 1625cm -1 The absorption peaks are respectively attributed to the stretching vibration of CH in the methyl group, the stretching vibration of C=O in the carboxyl group and the stretching vibration of C=O in the tertiary amide, indicating that captopril successfully reacts with the vinyl group in SHS-V-1 and is modified onto SHS-V-1.

[0032] The energy dispersive X-ray spectrum test results of SHS-CAP-1 are shown in Figure 4 (b). Figure 4 As can be seen in (b), peaks corresponding to carbon, oxygen, silicon, nitrogen and sulfur elements appeared in the spectrum, further indicating that captopril modification was successful.

[0033] 3. Application of the product as a hydrophilic interaction chromatography stationary phase material 1. Preparation of proteolysis samples 2 mg of immunoglobulin G was dissolved in 1 mL of ammonium bicarbonate solution containing 8 mol / L urea, pH=8.2, and a concentration of 100 mmol / L. Then, 80 µmol DL-1,4-dithiothreitol was added and the mixture was reacted at a constant temperature for 2 h in a 37°C water bath. Then, 40 µmol 2-iodoacetamide was added and the mixture was reacted in the dark for 40 min. Next, 7 mL of ammonium bicarbonate solution with a pH of 8.2 and a concentration of 100 mmol / L and 80 µg of trypsin were added and the mixture was enzymatically hydrolyzed in a 37°C water bath for 18 h. Finally, the mixture was desalted to obtain an immunoglobulin G enzymatic sample, which was packaged and freeze-dried and stored at -20°C for later use.

[0034] 2. Enrichment of N-glycopeptides in immunoglobulin G enzymatic hydrolysis samples using SHS-CAP-1 prepared in Example 1 2 mg of SHS-CAP-1 prepared in Example 1 was weighed into a centrifuge tube, equilibrated with 200 μL of loading solution (acetonitrile, water, trifluoroacetic acid mixed in a volume ratio of 85:14:1), then 200 μL of loading solution containing 9 μg of immunoglobulin G enzymatic hydrolyzed sample was added, and the mixture was shaken at 25°C and 1500 rpm for 30 min, then centrifuged, and the supernatant was discarded. Next, SHS-CAP-1 was washed three times with 200 μL of loading solution (purpose: to remove nonspecific adsorbed peptides), each time for 5 min, and then N-glycopeptides adsorbed on SHS-CAP-1 were eluted with 200 μL of elution solution (acetonitrile, water, trifluoroacetic acid mixed in a volume ratio of 30:69:1) for 20 min, centrifuged, and a portion of the eluate was directly analyzed by matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF Another portion of the eluate was lyophilized, deglycosylated using PNGase F using conventional techniques in the art, and then further analyzed by mass spectrometry using MALDI-TOF MS.

[0035] Among them, the method of mass spectrometry analysis using MALDI-TOF MS is: 0.5 μL of sample was dropped onto the MALDI target, and after it was naturally dried, 0.5 μL of 25 mg / mL 2,5-dihydroxybenzoic acid solution was covered on the sample point as a matrix. After it was completely naturally dried, it was sent to MALDI-TOF MS for mass spectrometry analysis.

[0036] The MALDI-TOF MS spectra of the IgG hydrolyzed sample before enrichment, the MALDI-TOF MS spectra after enrichment with SHS-CAP-1, and the MALDI-TOF MS spectra after sugar chain removal after enrichment are shown in Figure 2. Figure 5 .Depend on Figure 5 It can be seen that: (1) Before enrichment (a), a large number of highly abundant non-glycosylated peptide signals (mass-to-charge ratio below 2200) occupied most of the spectrum, and almost no N-glycopeptide signals (mass-to-charge ratio above 2200) were detected. (2) After using SHS-CAP-1 to enrich the IgG digested sample in a loading solution of acetonitrile, water, and trifluoroacetic acid (85 / 14 / 1, v / v / v) (b), the sample complexity was greatly reduced, the signal of the non-glycosylated peptide segment was almost completely removed, and the signal intensity of the N-glycopeptide was greatly increased. After mass-to-charge ratio comparison, 24 N-glycopeptides could be detected in 1.5 pmol of IgG digested sample after SHS-CAP-1 enrichment (Table 1). SHS-CAP-1 showed high specificity in N-glycopeptide enrichment. (3) After the enriched peptides were cleaved by PNGase F to remove the sugar chains (c), only two peaks appeared in the MALDI-TOF MS spectrum, with mass-to-charge ratios of 1158 and 1190, corresponding to the two deglycosylated modified peptides in the immunoglobulin G enzymatic hydrolysis sample, further confirming that SHS-CAP-1 has high specificity in N-glycopeptide enrichment.

[0037] Table 1 Detailed information of N-glycopeptides enriched from 1.5 pmol human IgG digested sample using SHS-CAP-1

[0038]

[0039] 3. Enrichment of N-glycopeptides in human serum using SHS-CAP-2 prepared in Example 2 In order to investigate the feasibility of using captopril-modified hollow nanosilica spheres for enriching N-glycopeptides in actual biological samples (e.g., human body fluids, tissues and cells; animal body fluids, tissues and cells), the SHS-CAP-2 prepared in Example 2 was used to enrich N-glycopeptides in 2 μL human serum and the LC-MS / MS results were used for N-glycosylation proteome analysis.

[0040] The enrichment process of N-glycopeptides in serum samples was similar to that of N-glycopeptides in immunoglobulin G enzymatic hydrolyzed samples. The difference was that the amount of SHS-CAP-2 was 5 mg, the amount of serum 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.

[0041] The results of N-glycosylation proteome analysis after three replicate enrichments of N-glycopeptides in 2 μL human serum samples using SHS-CAP-2 are shown in Figure 6 .Depend on Figure 6 It can be seen that after human serum samples were enriched by SHS-CAP-2, LC-MS / MS identified 314 specific N-glycosylated peptides and 165 specific N-glycosylation sites from 84 N-glycosylated proteins. Detailed information of the 84 N-glycosylated proteins and 314 specific N-glycosylated peptides is shown in Table 2.

[0042] Table 2 Detailed information of N-glycosylated proteins and specific N-glycosylated peptides enriched from 2 μL human serum samples using SHS-CAP-2.

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] 4. Enrichment of N-glycopeptides in human serum samples using the published HTC-Glc-10% MPC 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 human serum samples and the LC-MS / MS results were used for N-glycosylation proteome analysis.

[0055] The process of enriching N-glycopeptides in human serum samples using HTC-Glc-10% MPC is different from the previous process of enriching N-glycopeptides in human serum samples using SHS-CAP-2. The dosage of HTC-Glc-10% MPC is 10 mg, the loading time is 1 h, the elution time is 10 min each time, and the elution time is 40 min.

[0056] The results of N-glycosylation proteome analysis after three replicate enrichments of N-glycopeptides in 2 μL human serum samples using HTC-Glc-10% MPC are shown in Figure 7 .Depend on Figure 7 It can be seen that after HTC-Glc-10% MPC enrichment of human serum samples, LC-MS / MS identified 285 specific N-glycosylated peptides and 175 specific N-glycosylation sites from 92 N-glycosylated proteins.

[0057] contrast Figure 6 and Figure 7 It can be seen that the SHS-CAP-2 prepared by the present invention has a comparable N-glycopeptide enrichment effect to the published HTC-Glc-10%MPC, but the former requires less dosage and has a faster enrichment speed.

[0058] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing captopril-modified hollow nano-silicon spheres, characterized in that: Without adding any acid or base reagents, using water as solvent, F127 as template, mesitylene as micelle swelling agent, potassium sulfate as auxiliary agent, tetramethoxysilane and vinyltrimethoxysilane as silicon-based precursors, vinyl-modified hollow nano-silica spheres were prepared by sol-gel reaction; then, captopril was further modified on the hollow nano-silica spheres by thiol-ene photoinitiator reaction to obtain captopril-modified hollow nano-silica spheres.

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

3. The preparation method according to claim 1, characterized in that: The method for further modifying captopril on the hollow nano-silicon spheres by thiol-ene photoinitiator reaction is as follows: 0.12 g of vinyl-modified hollow nano-silica spheres were ultrasonically dispersed in 20 mL of a mixed solution of ethanol / water with a volume ratio of 1 / 1, and then 0.2 g of captopril and 0.2 g of benzoin dimethyl ether were added and dissolved by ultrasonic. After that, the mixture was photoinduced at a wavelength of 365 nm for 2 h, filtered, and the product was successively washed with water and ethanol, and dried under vacuum conditions to obtain captopril-modified hollow nano-silica spheres.

4. Captopril-modified hollow nano-silicon spheres prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the captopril-modified hollow nano-silica spheres as a hydrophilic interaction chromatography stationary phase material according to claim 4, characterized in that: Used to separate and enrich N-glycopeptides in biological samples.

6. The use according to claim 5, characterized in that: The biological sample is a body fluid sample, a tissue sample or a cell sample of a human or an animal.

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