Preparation method and application of strong hydrogen bond driven nitrogen-oxygen high-hydrophilic material
By using TEOS and 3AMPTS as silicon sources, polyamino nanosilicon spheres were prepared and processed by reverse phase microemulsion method to form hydrophilic materials rich in N→O groups, which solved the problem of insufficient hydrophilic interaction force in the prior art, achieved the enrichment of highly selective glycopeptides, and significantly improved the efficiency of glycoproteomics analysis.
Patent Information
- Application Number
- CN202411815962.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-11
AI Technical Summary
The existing hydrophilic chromatography is concentrated in glycoproteomics. Due to insufficient interaction force, the enrichment selectivity is not high, and the content of N→O groups in the existing materials is limited, which cannot meet the needs of highly hydrophilic materials.
Multiamino nanosilicon spheres were prepared by reverse phase microemulsion method using ethyl orthosilicate (TEOS) and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane (3AMPTS) as silicon sources, and polyamino nanosilicon spheres were prepared by the reverse phase microemulsion method, and treated with formaldehyde and hydrogen peroxide to form a hydrophilic material with rich and controllable N→O groups.
The prepared strong hydrogen bond-driven nitrogen and oxygen high hydrophilic materials have significantly improved hydrophilic interaction forces and can highly selectively enrich glycopeptides, including O-GlcNAc glycosylation, N-link glycosylation and O-link glycosylation peptides, significantly improving the efficiency of glycoproteomics analysis.
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Figure CN119588327A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a preparation method and application of a highly hydrophilic material, in particular to a preparation method and application of a strongly hydrogen-bonded driven nitrogen-oxygen highly hydrophilic material. Background Art
[0002] Protein glycosylation, as one of the most important post-translational modifications of proteins, is involved in regulating protein stability, activity, localization and interaction, and therefore plays an important role in cell physiology and disease processes (Mehboob MZ, Minglin L. Cell Death Dis. 2021, 12, 71). Glycoproteomics is mainly analyzed by mass spectrometry, but due to the low abundance, high heterogeneity, wide dynamic range and low ionization efficiency of glycosylated peptides, the analysis of glycoproteomics is extremely challenging (Zhonghan H, Wenqing G, Rong L, et al. Analyst. 2023, 149, 1090). Therefore, the development of efficient enrichment strategies is crucial for in-depth research in glycoproteomics.
[0003] Hydrophilic interaction chromatography is an important glycopeptide enrichment method. This method has the advantages of intact sugar chain structure retention, high reproducibility, and improved mass spectrometry ionization efficiency. It is widely used in glycoproteomics enrichment (Jing J, Wangtao Y, Xiaohong Q, Chinese J. Anal. Chem. 2014, 2, 159). However, the main challenge of this method is that the interaction between it and glycopeptides is relatively weak, resulting in insufficient enrichment selectivity. In order to solve this problem, it is urgent to develop highly hydrophilic materials. Hydrophilic interaction forces mainly include hydrophilic distribution, hydrogen bonding, and electrostatic interactions (Qing G, Yan J, He X, et al. Trends Anal. Chem. 2020, 124, 115570). Among them, hydrogen bonding is one of the most important driving forces for retaining and separating polar / hydrophilic compounds (including glycosides and carbohydrates). In recent years, researchers have discovered that N→O groups can form strong hydrogen bond interactions with proton donors such as −OH groups as proton acceptors (Luo Y, Ma H, Zhang S, Zheng D, Che P, Liu X, Zhang M, Gao J, Xu J, J. Am. Chem. Soc. 2020, 142, 13, 6085). Based on this principle, silica materials with surface modified with N→O groups have been synthesized and successfully used for the enrichment of monosaccharide-modified O-GlcNAc glycopeptides in pancreatic cancer cells (Wu C, Shi S, Hou C, Luo Y, Byers S, Ma J, ACS Appl. Mater. Interfaces. 2022, 14, 47482). However, it only modifies the surface of silica gel with a single layer of functional groups, and the content of the prepared N→O groups is limited, resulting in insufficient hydrophilic interaction and low enrichment specificity for the target.
[0004] The reverse microemulsion method is one of the important methods for preparing organic-inorganic hybrid silica gel materials. Its water core is like a "microreactor". The surface of the nanoparticles generated in the "microreactor" is coated with a layer of surfactant molecules, which makes it difficult for the particles to aggregate. The added silicon source can diffuse from the oil phase into the water core to undergo hydrolysis and condensation reactions. By controlling the content of the organic silicon source, it is beneficial to control the content of functional groups in the silica gel material. However, there has been no report on the successful preparation of hydrophilic materials with rich and controllable N→O groups using the reverse microemulsion method. Summary of the invention
[0005] The present invention aims to solve the technical problems that the content of N→O groups in the prior art is limited, resulting in insufficient hydrophilic interaction and low enrichment specificity for target substances, and provides a preparation method and application of a strong hydrogen bond driven nitrogen-oxygen highly hydrophilic material.
[0006] The technical solution of the present invention is: a method for preparing a strong hydrogen bond driven nitrogen oxide highly hydrophilic material, characterized in that tetraethyl orthosilicate (TEOS) and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane (3AMPTS) are used as silicon sources, and a reverse microemulsion method is used to prepare polyamino nano-silicon balls with uniform particle size and controllable functional group content, and then formaldehyde (HCHO) is used to dimethylate the amino groups on the polyamino nano-silicon balls, and hydrogen peroxide (H2O2) is used to oxidize them to generate N→O groups.
[0007] Preferably, the following steps are followed: Step 1. Prepare solution A using a surfactant, a co-surfactant and an oil phase, prepare solution B using an alkaline catalyst and water, add solution B into solution A and mix well to obtain a microemulsion; Step 2. Under vigorous stirring, add tetraethyl orthosilicate (TEOS) and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane (3AMPTS) to the microemulsion and continue stirring, wherein the 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane accounts for 10-90% of the total mass of tetraethyl orthosilicate and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane, and after the stirring is completed, add ethanol demulsifier, centrifuge, reflux with ethanol to remove the surfactant, disperse in ethanol, and centrifuge and dry to obtain polyamino nanosilicon spheres (3AMPTS-SiO2NPS); Step 3. Add a phosphate buffer solution with a pH of 4-11 to the prepared polyamino nano-silicon spheres (3AMPTS-SiO2 NPS) and stir evenly, wherein the nano-silicon spheres account for 0.5-3% of the total mass; add formaldehyde (HCHO) to a final concentration of 0.5 - 3 mol / L), add sodium cyanoborohydride (NaBH3CN) to a final concentration of 0.5 - 2 mol / L, mix and stir for 12 - 36 h to obtain dimethyl-modified silica spheres (Dimethyl-3AMPTS-SiO2NPS); Step 4. Suspend the dimethyl modified silica spheres in H2O2 solution, the amount of dimethyl modified silica spheres added accounts for 5-15% of the total mass, stir overnight at 40-100 ℃, then centrifuge at 2000-8000 rpm for 1-10 min, remove the supernatant; then add H2O2 solution, the molar ratio of which is 0.5-2 to the first added H2O2 solution, mechanically stir at 40-100 ℃ for 1-12 h, centrifuge, and dry to obtain a hydrophilic material (NO-3AMPTS-SiO2 NPS).
[0008] Preferably, the surfactant is a nonylphenol polyoxyethylene ether series (NP-5, NP-7, NP-9, etc.), sodium di(2-ethylhexyl) succinate sulfonate (AOT) or Triton X-100, and the molar percentage of the amount used in the microemulsion is 1-5%; the co-surfactant is n-propanol, n-butanol or n-hexanol, and the molar percentage of the amount used in the microemulsion is 5-20%; the oil phase is cyclohexane, n-hexane or toluene, and the molar percentage of the amount used in the microemulsion is 20-40%; the alkaline catalyst is ammonia water, ethylenediamine, diethylamine, triethylamine or sodium hydroxide, and the molar percentage of the amount used in the microemulsion is 1-5%.
[0009] A method for preparing a strong hydrogen bond driven nitrogen oxide highly hydrophilic material according to claim 1 and its application in enriching glycoproteins in biological samples.
[0010] The specific method of preferred application is to dissolve the enzymatic hydrolysis product of the biological sample in a loading buffer to a final concentration of 0.1-10 mg / mL, mix with 1-50% by mass fraction of a strong hydrogen bond driven nitrogen oxide high hydrophilic material and incubate for 0.5-5 h, wash the strong hydrogen bond driven nitrogen oxide high hydrophilic material with a loading buffer washing solution, and finally elute with an eluent, collect the eluent and freeze-dry it to obtain enriched glycopeptides; The loading buffer is a mixed solution composed of acetonitrile (ACN), H2O and ion additives, wherein the ion additives are formic acid, acetic acid, ammonium formate or ammonium bicarbonate, accounting for 0-40% of the mass fraction of the loading buffer. The volume ratio of acetonitrile (ACN) to the loading buffer is 60-95%. The eluent is a mixed solution composed of acetonitrile (ACN), H2O and ion additives, wherein the ion additives are formic acid, acetic acid, ammonium formate or ammonium bicarbonate, accounting for 0-40% of the mass fraction of the eluent, and acetonitrile accounts for 0-60% of the mass fraction of the eluent.
[0011] The present invention uses tetraethyl orthosilicate (TEOS) and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane (3AMPTS) as silicon sources, and adopts the reverse microemulsion method to synthesize a polyamine-based silica sphere matrix in one step, and utilizes the Mannich reaction between formaldehyde and amino groups to dimethylate the amine groups in the material, and finally uses hydrogen peroxide oxidation to convert dimethyl tertiary amine into N→O groups, successfully preparing a hydrophilic material with rich N→O groups and controllable hydrophilicity, and applying it to the enrichment of glycopeptides. By controlling the feed ratio of the organic silicon source, the content of N→O functional groups in the material can be regulated, and the hydrophilicity of the material can be further improved, so as to achieve highly selective enrichment of all types of glycopeptides, including O-GlcNAc glycosylation, N-link glycosylation, and O-link glycosylation peptides. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 This is the zeta potential diagram of the strong hydrogen bond driven nitrogen and oxygen highly hydrophilic material prepared in Example 1 of the present invention.
[0013] Figure 2 This is a SEM image of the strongly hydrogen-bonded driven nitrogen-oxygen highly hydrophilic material prepared in Example 1 of the present invention.
[0014] Figure 3 This is a TEM image of the strongly hydrogen-bonded driven nitrogen-oxygen highly hydrophilic material prepared in Example 1 of the present invention.
[0015] Figure 4 This is the mass spectrum of Example 1 of the present invention applied to the enrichment of sugar N-link glycopeptides in IgG. DETAILED DESCRIPTION Example 1
[0016] The method for preparing the strong hydrogen bond driven nitrogen-oxygen highly hydrophilic material of the present invention is carried out according to the following steps: Step 1. First, 15.11 g of surfactant NP-7, 8 g of co-surfactant n-butanol and 31.55 g of organic phase cyclohexane were uniformly mixed to obtain solution A, and then 2 g of ammonia water and 5.5 g of water were added to prepare solution B, and solution B was added to solution A and mixed uniformly to obtain a microemulsion; Step 2. Under vigorous stirring, add 4.1665 g of tetraethyl orthosilicate (TEOS) and 1.3271 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane (3AMPTS) to the microemulsion and continue stirring. After stirring, add ethanol demulsifier, centrifuge, reflux with ethanol to remove the surfactant, disperse in ethanol, and centrifuge and dry to obtain polyamino nanosilicon spheres (3AMPTS-SiO2NPS); Step 3. Add a phosphate buffer solution with a pH of 4-11 to the prepared polyamino nano-silicon spheres 3AMPTS-SiO2 NPS and stir evenly, wherein the nano-silicon spheres account for 1% of the total mass; add formaldehyde (HCHO) to a final concentration of 1 mol / L, add sodium cyanoborohydride (NaBH3CN) to a final concentration of 1 mol / L, mix and stir for 24 h to obtain dimethyl-modified silica spheres (Dimethyl-3AMPTS-SiO2NPS); Step 4. Suspend dimethyl-modified silica spheres (Dimethyl-3AMPTS-SiO2NPS) in H2O2 solution, the amount of dimethyl-modified silica spheres added accounts for 10% of the total mass, stir overnight at 40-100 °C, then centrifuge at 8000 rpm for 5 minutes, remove the supernatant; then add H2O2 solution, the molar ratio of which is 1 to the first added H2O2 solution, mechanically stir at 70 °C for 12 hours, centrifuge, and dry to obtain a hydrophilic material (NO-3AMPTS-SiO2 NPS). Example 2
[0017] The difference from Example 1 is that 0.6636 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a highly hydrophilic nitrogen-oxygen material with a functional group content of 10%. Example 3
[0018] The difference from Example 1 is that 1.9907 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a highly hydrophilic nitrogen-oxygen material with a functional group content of 30%. Example 4
[0019] The difference from Example 1 is that 2.6542 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a highly hydrophilic nitrogen-oxygen material with a functional group content of 40%. Example 5
[0020] The difference from Example 1 is that 3.3178 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a highly hydrophilic nitrogen-oxygen material with a functional group content of 50%. Example 6
[0021] The difference from Example 1 is that 3.9813 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a highly hydrophilic nitrogen-oxygen material with a functional group content of 60%. Example 7
[0022] The difference from Example 1 is that 4.6449 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a 70% strong hydrogen bond driven nitrogen and oxygen highly hydrophilic material. Example 8
[0023] The difference from Example 1 is that 5.3084 g of 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane is added, and the rest is the same as Example 1, to obtain a highly hydrophilic nitrogen-oxygen material with a functional group content of 80%.
[0024] experiment: The Zeta potential of the hydrophilic material obtained in Example 1 was measured to determine the surface charge of the material. Figure 1 As shown. According to the DLVO (Derjaguin-Landau-Verwey-Overbee) theory, stable colloidal dispersion can be produced in the density range greater than +30 mV and less than -30 mV. It was found that SiO2 has a negative surface charge and shows good stability between pH = 5.8 - 8.0; 3AMPTS-SiO2 and Dimethyl-SiO2 have a positive surface charge and have high stability between pH = 5.0-8.0. The NO-3AMPTS-SiO2 NPS (NO-SiO2) of Example 1 of the present invention loses its positive surface charge rapidly with the increase of alkalinity, has an equipotential point at pH = 5.5, and then has a negative surface charge on the surface.
[0025] 2. The scanning electron microscope image of the strong hydrogen bond driven nitrogen and oxygen highly hydrophilic material NO-3AMPTS-SiO2 NPS prepared in Example 1 of the present invention is as follows: Figure 2 As shown in the transmission electron microscopy image Figure 3 As shown, from Figure 2 , Figure 3 It can be seen that the hydrophilic material obtained in Example 1 of the present invention has a granular morphology and is uniform in size.
[0026] 3. The NO-3AMPTS-SiO2 NPS prepared in Example 1 was used for the enrichment experiment of N-link glycosylated peptides in IgG enzymatic hydrolysate, and different gradient ACN concentrations were designed to determine the effect of ACN ratio on the enrichment of glycopeptides. The specific method of the experiment is to dissolve the enzymatic hydrolysate of the biological sample in a loading buffer to a final concentration of 10 mg / mL, mix with a 20% mass fraction of a strong hydrogen bond driven nitrogen oxide high hydrophilic material and incubate for 1 h, wash the strong hydrogen bond driven nitrogen oxide high hydrophilic material with a loading buffer washing solution, and finally elute with an eluent, collect the eluent and freeze-dry it to obtain enriched glycopeptides; The loading buffer is a mixed solution composed of acetonitrile (ACN), H2O and ion additives, wherein the ion additives are formic acid, acetic acid, ammonium formate or ammonium bicarbonate, accounting for 10% of the mass fraction of the loading buffer. The volume ratio of acetonitrile (ACN) to the loading buffer is 80%. The eluent is a mixed solution composed of acetonitrile (ACN), H2O and ion additives, wherein the ion additives are formic acid, acetic acid, ammonium formate or ammonium bicarbonate, accounting for 10% of the mass fraction of the eluent, and acetonitrile accounts for 30% of the mass fraction of the eluent.
[0027] The experiment was identified by MALDI-TOF-MS, and the results were as follows Figure 4 As shown. Figure 4 It can be seen that in the unenriched IgG hydrolysate, the glycopeptide signal was suppressed by the non-glycopeptide signal, and only 3 glycopeptide signals could be detected; while in Example 1 of the present invention, up to 26 glycopeptides could be detected under the enrichment condition of 75% ACN, proving that NO-3AMPTS-SiO2 NPS has good enrichment selectivity for N-link glycopeptides.
[0028] The NO-3AMPTS-SiO2NPS prepared in Example 1 was used to enrich the O-GlcNAc glycosylated peptides in the pancreatic cancer cell enzymatic hydrolysis sample, and LC-MS (Thermo, Lumos Fusion) was used for identification. One injection can detect 342 O-GlcNAc glycoproteins and 698 O-GlcNAc glycosylation sites. It shows that the NO-3AMPTS-SiO2NPS prepared by the present invention has good enrichment selectivity for O-GlcNAc glycopeptides.
Claims
1. A method for preparing a strong hydrogen bond driven nitrogen-oxygen highly hydrophilic material, characterized in that Polyamino nano-silica spheres with uniform particle size and controllable functional group content are prepared by using ethyl orthosilicate and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane as silicon sources through a reverse microemulsion method. The amino groups on the polyamino nano-silica spheres are dimethylated with formaldehyde and oxidized with hydrogen peroxide to generate N→O groups.
2. The method for preparing the strong hydrogen bond driven nitrogen-oxygen highly hydrophilic material according to claim 1, characterized in that Follow these steps: Step 1. Prepare solution A using a surfactant, a co-surfactant and an oil phase, prepare solution B using an alkaline catalyst and water, add solution B into solution A and mix well to obtain a microemulsion; Step 2. Under vigorous stirring, add ethyl orthosilicate and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane to the microemulsion and continue to stir vigorously, wherein the 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane accounts for 10-90% of the total mass of ethyl orthosilicate and 3-(2-(2-aminoethylamino)ethylamino)propyl-trimethoxysilane. After the stirring is completed, add ethanol demulsifier, centrifuge, reflux with ethanol to remove the surfactant, disperse in ethanol, and centrifuge and dry to obtain polyamino nano-silicon spheres; Step 3. Add a phosphate buffer solution with a pH of 4-11 to the prepared polyamino nano-silicon spheres and stir evenly, wherein the nano-silicon spheres account for 0.5-3% of the total mass; add formaldehyde to a final concentration of 0.5-3 mol / L, add sodium cyanoborohydride to a final concentration of 0.5-2 mol / L, mix and stir for 12 - 36 h to obtain dimethyl-modified silica spheres (Dimethyl-3AMPTS-SiO2NPS); Step 4. Suspend the dimethyl modified silica spheres in the H2O2 solution, the amount of the dimethyl modified silica spheres added is 5-15% of the total mass, stir overnight at 40-100 ℃, then centrifuge at 2000-8000 rpm for 1-10 min, remove the supernatant; then add the H2O2 solution, the molar ratio of which is 0.5-2 to the first added H2O2 solution, mechanically stir at 40-100 ℃ for 1-12 h, centrifuge, and dry to obtain a hydrophilic material.
3. The method for preparing the strong hydrogen bond driven nitrogen and oxygen highly hydrophilic material according to claim 2, characterized in that: The surfactant is a nonylphenol polyoxyethylene ether series (NP-5, NP-7, NP-9, etc.), sodium di(2-ethylhexyl) succinate sulfonate or Triton X-100, and the molar percentage of the amount used in the microemulsion is 1-5%; the co-surfactant is n-propanol, n-butanol or n-hexanol, and the molar percentage of the amount used in the microemulsion is 5-20%; the oil phase is cyclohexane, n-hexane or toluene, and the molar percentage of the amount used in the microemulsion is 20-40%; the alkaline catalyst is ammonia water, ethylenediamine, diethylamine, triethylamine or sodium hydroxide, and the molar percentage of the amount used in the microemulsion is 1-5%.
4. Use of the strong hydrogen bond driven nitrogen oxide highly hydrophilic material prepared by the method as claimed in claim 1 in enriching glycoproteins in biological samples.
5. The use of the strong hydrogen bond driven nitrogen oxide highly hydrophilic material according to claim 4 in the enrichment of glycoproteins in biological samples, characterized in that The application method is to dissolve the enzymatic hydrolysis product of the biological sample in a loading buffer to a final concentration of 0.1-10 mg / mL, mix with a 1-50% mass fraction of a strong hydrogen bond driven nitrogen oxide high hydrophilic material and incubate for 0.5-5 h, wash the strong hydrogen bond driven nitrogen oxide high hydrophilic material with a loading buffer washing solution, and finally elute with an eluent, collect the eluent and freeze-dry it to obtain enriched glycopeptides; The loading buffer is a mixed solution composed of acetonitrile, H2O and ion additives, wherein the ion additives are formic acid, acetic acid, ammonium formate or ammonium bicarbonate, accounting for 0-40% of the mass fraction of the loading buffer. The volume ratio of acetonitrile to the loading buffer is 60-95%. The eluent is a mixed solution composed of acetonitrile (ACN), H2O and ion additives, wherein the ion additives are formic acid, acetic acid, ammonium formate or ammonium bicarbonate, accounting for 0-40% of the mass fraction of the eluent, and acetonitrile accounts for 0-60% of the mass fraction of the eluent.