A method for the preparation of a material enriched in hydrophilic biological substances

Abstract

The application discloses a preparation method of a material for enriching hydrophilic biological substances, and the core of the method is to perform peptide segment enrichment through specific binding of chemically modified magnetic nanoparticles and glycosylated peptide segments, so that the problems of high cost, complicated operation and limited identification throughput are solved, and an efficient, stable and easy-to-operate technical process is provided for glycoproteomics research. High selectivity: the surface of the magnetic beads is a combination of mesoporous structures and quaternary ammonium salt groups, which significantly improves the selectivity of the glycosylated peptide segments and reduces non-specific adsorption. The number of N-glycans can be identified to an average of 2733, and the number of O-glycans can be identified to an average of 1777. Simple operation: the introduction of the magnetic beads simplifies the enrichment process and facilitates subsequent automated experiments and large-scale applications. Cost reduction: by simplifying the synthesis method and improving the stability of the material, the preparation and application cost of the material is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation method of a material for enriching hydrophilic biological substances. BACKGROUND

[0002] Glycoproteins play an indispensable role in a variety of biological processes such as signal transduction, cell adhesion, and protein folding. Therefore, the abnormal expression of glycoproteins is closely related to the occurrence and development of a variety of diseases. Among the cancer biomarkers used in medical diagnosis, more than two-thirds are glycoproteins, including mucin epitopes (CA15-3) and alpha-fetoprotein (CA-125). Due to the complexity of protein glycosylation, such as glycan structure heterogeneity and glycosylation site diversity, traditional bulk analysis methods are difficult to accurately analyze, and therefore a bottom-up glycoproteomics method is needed to obtain higher glycopeptide resolution and glycosylation site specificity by digesting glycoproteins, enriching glycopeptides, and combining mass spectrometry analysis. In specific experiments, the proportion of glycopeptides is relatively low compared to the total content of the obtained peptides, and the mass spectrometry signal of glycopeptides may be interfered or masked by other peptides. Therefore, there is an urgent need for new methods to improve the detection rate of glycopeptides.

[0003] Hydrophilic interaction chromatography (HILIC) technology has attracted much attention in the field of glycopeptide enrichment in recent years. Its core mechanism is to utilize the high hydrophilicity of the sugar chains in glycopeptides to achieve separation from other non-glycosylated peptides by distributing between the hydrophilic stationary phase and the organic phase-water phase mobile phase. Zwitterionic HILIC (ZIC-HILIC) material, as an important branch of HILIC technology, has attracted much attention due to its unique charge balance characteristics. ZIC-HILIC material contains both cationic and anionic groups on its surface, which can form a highly hydrated layer through electrostatic interaction with water molecules while maintaining overall electrical neutrality. This hydration layer not only enhances the hydrophilicity of the material, but also provides multiple action sites for glycopeptide enrichment, including hydrogen bonding, electrostatic interaction, and hydrophilic interaction. This multiple action mechanism enables ZIC-HILIC material to exhibit excellent glycopeptide enrichment capacity in complex biological samples, especially when dealing with peptides containing multiple post-translational modifications, it can achieve high selectivity and high recovery rate.

[0004] However, despite the theoretical advantages of ZIC-HILIC materials, their practical application still faces many challenges. First, the preparation process of traditional ZIC-HILIC materials is complex, often requiring multiple chemical reactions and purification steps, which not only increases the preparation cost, but also may cause performance differences between batches of materials. Second, ZIC-HILIC materials in actual operation usually rely on centrifugation or filtration and other separation means, which are prone to cause loss of target substances when dealing with trace samples, and are difficult to realize high-throughput operation. In addition, the enrichment efficiency of existing ZIC-HILIC materials in complex biological samples (such as serum, urine or tissue lysate) still needs to be improved, especially in the capture of low-abundance glycopeptides, which often fails to meet the needs of clinical proteomics research.

[0005] These technological bottlenecks have prompted researchers to continuously explore new material designs and enrichment strategies. In 2017, Weiming Yang's team studied the enrichment of N / O-glycopeptides in serum samples using ZIC-HILIC material, a commercial product of The Nest Group, identifying 313 N-glycopeptides and 45 O-glycopeptides. While adding an experimental step to remove N-glycopeptides before enrichment significantly increased the number of identified O-glycopeptides by 66, the identification throughput remained low and the operational complexity increased (Weiming Yang, et al. Analytical Chemistry, 2017, 89 (21), 11193-11197). In 2021, Yiju Chen's team used ZIC-cHILIC material for glycosylation enrichment experiments. Through the synergistic effect of zwitterionic ligands and dodecyl groups, the specificity of glycopeptide enrichment was increased to over 90%, with a single enrichment identification throughput of 1369 glycopeptides. However, its reliance on centrifugation limits its application in high-throughput clinical sample analysis (Yiju Chen, et al. Analytical Chemistry, 2021, 93 (48), 15931-15940). In 2022, Linhua Yi's team proposed superhydrophilic Fe3O4-CG nanospheres, which simplified the ZIC-HILIC material preparation process through a one-step hydrothermal method. However, this method only identified 131 glycosylated peptides in healthy human samples and 180 glycosylated peptides in Alzheimer's disease samples. Although the material preparation process was simplified, its identification throughput in complex disease samples remained low (Linhua Yi, et al. Journal of Chromatography A, 2022, 1669, 462929). These studies indicate that the current ZIC-HILIC technology still faces three major challenges: first, the contradiction between material preparation process and performance indicators, where simplifying the preparation process may come at the cost of sacrificing the number of enriched peptides; second, the insufficient automation of the operation process, which restricts the feasibility of clinical translation; and third, the ability to enrich low-abundance glycopeptides needs to be improved. Summary of the Invention

[0006] In order to solve the problems of low binding efficiency, high synthesis cost and complex operation steps of glycosylated peptide segments, a magnetic nanocomposite material is invented for glycosylated peptide segment enrichment, and proteomics analysis is carried out. First, Fe3O4 nanoparticles are synthesized by co-precipitation method, and the stability and surface modifiability of the nanoparticles are improved by SiO2 coating. Then, the mesoporous SiO2 shell is constructed by ATMS and CTAB template method, and the hydrophilic polymer layer (MBAAm-co-SBMA) is introduced on the surface of the mesoporous SiO2 shell by free radical polymerization method, so as to give the material good glycopeptide enrichment ability. The mouse brain tissue is used in the experiment, and after lysis, enzymatic hydrolysis and desalting treatment, the synthesized magnetic beads are used for glycopeptide enrichment. Through magnetic separation, washing and gradient elution, glycosylated peptides with high purity are obtained. The method is simple in operation and high in enrichment efficiency, and provides a low-cost and efficient solution for glycoproteomics research. In addition, the material has good batch preparation potential and can be compatible with automatic sample processing system, providing reliable support for high-throughput glycosylation modification analysis.

[0007] The application provides a preparation method of a magnetic nanoparticle for specifically separating glycosylated peptides, and the method comprises the following steps:

[0008] 1) Fe3O4@SiO2 nanoparticles are dispersed in an ethanol concentrated ammonia solution, CTAB is added, TEOS and allyltrimethoxysilane are slowly added dropwise under vigorous stirring, after the reaction is completed, the allyl-functionalized mesoporous SiO2 coated magnetic particles are separated by a magnet, washed with ethanol and water alternately to remove CTAB and unreacted monomers, and dried in a 50 ℃ oven to obtain Fe3O4@SiO2@a-mSi;

[0009] 2) The dried allyl-functionalized mesoporous SiO2 coated magnetic particles, SBMA and MBAAm are added to an ACN solution, dissolved oxygen is removed, AIBN is added, and the free radical polymerization reaction is carried out by heating to 75 ℃, 5-60% volume ACN is distilled out by slowly increasing the temperature from 75 ℃ to 100 ℃, the temperature is cooled to room temperature and stirring is carried out, the nanoparticles are washed with ACN to remove unreacted monomers and oligomers, and dried at 50 ℃ to obtain the final Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA).

[0010] Further, 35% volume ACN is distilled out;

[0011] Preferably, the components of the ethanol concentrated ammonia solution are distilled water, ethanol and concentrated ammonia solution, and the volume ratio is 15-23:3-8:0.5-2;

[0012] Preferably, the components of the ethanol concentrated ammonia solution are distilled water, ethanol and concentrated ammonia solution, and the volume ratio is 20:5:1.

[0013] In the present application, the concentrated ammonia solution refers to a 32% concentrated ammonia aqueous solution.

[0014] Further, the ratio of the Fe3O4@SiO2 nanoparticles, the ethanol concentrated ammonia solution, CTAB, TEOS, and allyltrimethoxysilane is 0.5-2 g: 240-300 mL: 0.5-8 g: 5-40 mL: 1-20 g.

[0015] Preferably, the ratio of the Fe3O4@SiO2 nanoparticles, the ethanol concentrated ammonia solution, CTAB, TEOS, and allyltrimethoxysilane is 1 g: 260 mL: 3 g: 23.5 mL: 5 g.

[0016] Further, the ratio of the allyl-functionalized mesoporous SiO2-coated magnetic particles, SBMA, MBAAm, and ACN solution in step 2) is 0.01-1 mg: 0.1-100 g: 0.1-100 g: 20-500 mL.

[0017] Preferably, the ratio of the allyl-functionalized mesoporous SiO2-coated magnetic particles, SBMA, MBAAm, and ACN solution in step 2) is 40 mg: 0.2 g: 1 g: 100 mL.

[0018] Further, the ratio of the AIBN as an initiator and the ACN solution is 0.01-5 g: 20-500 mL.

[0019] Preferably, the ratio of the AIBN as an initiator and the ACN solution is 0.0225 g: 100 mL.

[0020] Further, the Fe3O4@SiO2 nanoparticles are synthesized by the following method:

[0021] The Fe3O4 nanoparticles are dispersed in a mixed solution of distilled water, ethanol, and 32% concentrated ammonia, and TEOS is slowly added dropwise under vigorous stirring. The SiO2 is uniformly coated on the surface of the Fe3O4 by continuing to stir. After the reaction is completed, the silica-coated Fe3O4 nanoparticles are separated by a magnet and washed with ethanol and deionized water alternately to remove unreacted TEOS and byproducts.

[0022] Further, the mixed ratio of the distilled water, ethanol, and 32% concentrated ammonia is 1-100: 5: 0.1-5.

[0023] Preferably, the mixed ratio of the distilled water, ethanol, and 32% concentrated ammonia is 20: 5: 1.

[0024] Further, the ratio of the Fe3O4 nanoparticles to the mixed solution is 0.2-2 g: 100-500 mL;

[0025] Preferably, the ratio of the Fe3O4 nanoparticles to the mixed solution is 1 g: 260 mL.

[0026] Further, the ratio of the Fe3O4 nanoparticles to TEOS is 0.3-3 g: 0.3-0.7 g;

[0027] Preferably, the ratio of the Fe3O4 nanoparticles to TEOS is 1 g: 0.5 g.

[0028] Further, the Fe3O4 nanoparticles are synthesized by the following method:

[0029] FeCl3·6H2O and FeCl2·4H2O are dissolved in deionized water, degassed with nitrogen, the solution is heated to 80°C, then 32% NH4OH solution is slowly added dropwise, the reaction is maintained, the solid is separated by a magnet, finally, 0.1 mol / L NaCl solution is used for washing to remove unreacted ions and byproducts, and the Fe3O4 nanoparticles are dried in a 50°C oven.

[0030] Further, the ratio of the deionized water to FeCl3·6H2O, FeCl2·4H2O, 32% NH4OH solution is 50-300 mL: 1-30 g: 1-30 g: 8-20 mL;

[0031] Preferably, the ratio of the deionized water to FeCl3·6H2O, FeCl2·4H2O, 32% NH4OH solution is 100 mL: 10.4 g: 4 g: 15 mL.

[0032] In some embodiments, the method for specifically separating glycosylated peptide fragments magnetic nanoparticles is prepared by synthesizing Fe3O4 nanoparticles by co-precipitation method; Fe3O4 is coated with SiO2 on the surface of Fe3O4 by using tetraethyl orthosilicate to form Fe3O4@SiO2 particles; Fe3O4 magnetic nanoparticles coated with allyl functionalized mesoporous silica (Fe3O4@SiO2@a-mSi) are synthesized by combining sol-gel method (Sol-Gel method) with self-assembly method; a poly(MBAAm-co-SBMA) coating layer is synthesized on the surface of Fe3O4@SiO2@a-mSi nanoparticles by free radical polymerization method (Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA)). The poly(MBAAm-co-SBMA) magnetic nanoparticles are used for glycosylated peptide fragment enrichment and proteomics analysis.

[0033] The term "TEOS" used in the present invention refers to Tetraethyl orthosilicate, chemical formula Si(OC2H5)4, which is an important organosilicon compound, colorless transparent liquid at room temperature, with slight volatility and a weak odor similar to ethanol. Its molecular structure is centered on a silicon atom, connected to four oxygen atoms, respectively, to methoxy groups (-OC2H5), forming a highly symmetrical tetrahedral configuration. As a precursor of silicon dioxide (SiO2), TEOS has a wide range of applications in materials science and industry.

[0034] The term "CTAB" used in the present invention refers to Cetyltrimethylammonium bromide, a typical cationic quaternary ammonium salt surfactant. Its molecular structure consists of a long-chain cetyl hydrophobic tail, a positively charged trimethylammonium hydrophilic head, and a bromide counterion. This amphiphilicity allows it to significantly reduce the surface tension of the solution and form micelles in water. When the concentration of CTAB exceeds the critical micelle concentration (CMC), the hydrophobic chains aggregate inward, and the hydrophilic heads arrange outward, forming spherical or rod-shaped micelles. This property makes it play a key role in the synthesis of nanomaterials.

[0035] The term "Allyltrimethoxysilane (ATMS)" used in the present invention refers to a functional silane coupling agent with both organic and inorganic properties. Its molecular structure is centered on a silicon atom, with three hydrolyzable methoxy groups (-OCH3) on one side and an allyl group (CH2=CH-CH2-) bonded through a silicon-carbon bond on the other side. This dual functional design gives it unique reactivity. ATMS is a colorless transparent liquid at room temperature, with slight volatility, and is soluble in organic solvents such as ethanol and acetone. Its core chemical properties are reflected in two types of reactions: one is the hydrolysis of methoxy groups under acidic or basic conditions to form silanol (Si-OH), which then forms a three-dimensional network structure through condensation reaction; the other is the carbon-carbon double bond in the allyl group, which can participate in free radical polymerization, addition or crosslinking reaction, thereby forming chemical bonding with organic polymer materials (such as polyethylene, rubber, resin, etc.). Based on this dual reaction mechanism, ATMS is widely used as an interfacial modifier, which can bridge inorganic materials (such as glass fibers, metal oxides, fillers) and organic polymers, significantly improving the mechanical strength, water resistance and heat resistance of the composite materials.

[0036] The term "Sulfobetaine methacrylate (SBMA)" used in the present invention refers to a typical zwitterionic functional monomer, with both positive and negative charge groups, with excellent hydrophilicity, antifouling property and biocompatibility. Its molecular structure consists of three parts: a methacrylate group (providing a polymerizable double bond), a sulfonate anion (-SO3 - ) and a quaternary ammonium salt cation (-N+ (CH3)2), through chemical bonds to form intramolecular charge self-balanced zwitterion structure. This unique charge distribution makes it exhibit "anti-polyelectrolyte effect" in aqueous solution, that is, it can still maintain solubility in high ionic strength environment, avoiding precipitation due to charge shielding.

[0037] The term "N,N'-methylenebisacrylamide (MBAAm)" used in the present application is a bifunctional crosslinking agent, widely used in polymer network construction, especially in the synthesis of hydrogel and polyacrylamide materials. Its molecular structure is bridged by methylene (-CH2 - ) to two acrylamide groups, each containing a carbon-carbon double bond (CH2=CH-CONH2) that can participate in free radical polymerization. This property enables it to connect two or more polymer chains simultaneously in the polymerization reaction, forming a stable three-dimensional cross-linked network, thereby significantly improving the mechanical strength, chemical stability and swelling performance of the material. MBAAm has high water solubility (especially in warm water), mild reaction conditions (usually used with ammonium persulfate initiator), and crosslinking density can be precisely controlled by its feed ratio.

[0038] The term "azobisisobutyronitrile (AIBN)" used in the present application is a highly efficient free radical initiator, widely used in the field of high polymer polymerization and organic synthesis. Its molecular structure contains two isobutyronitrile groups (-C(CH3)2CN) connected by azo group (-N=N-), this high-energy bond is easy to break under heating (60-80 ℃) or light, decomposing into nitrogen (N2) and two isobutyronitrile free radicals (·C(CH3)2CN), which can initiate free radical chain polymerization of monomers (such as acrylate, styrene, acrylonitrile, etc.). AIBN is a white crystalline powder, slightly soluble in water, easily soluble in organic solvents such as acetone and toluene, has heat and light sensitivity, and needs to be stored in the dark, low temperature (<20 ℃) and dry to prevent spontaneous decomposition. As a "trigger" for polymerization, its half-life is closely related to temperature (such as 64 ℃, half-life about 10 hours), which can precisely control the reaction rate and molecular weight distribution by adjusting the temperature.

[0039] The term "ACN" used in the present application refers to acetonitrile.

[0040] The term "SDC" used in the present application refers to sodium dodecyl sulfate.

[0041] The term "Fe3O4@SiO2@a-mSi" used in the present application refers to allyl functionalized Fe3O4@SiO2.

[0042] The term "FA" used in the present application refers to formic acid, which is a colorless, strong polar, weak acidic (pKa≈3.75) and highly volatile organic acid, widely used in the mobile phase system of liquid chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS).

[0043] The term "TFA" used in the present application refers to trifluoroacetic acid, molecular formula CF3COOH, molecular weight 114.02 g / mol, which is a strong organic acid, stronger than acetic acid. It is a commonly used additive in liquid chromatography, mainly used to adjust the pH of the mobile phase, improve peak shape and separation effect.

[0044] The present application provides a Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) prepared by the above-mentioned preparation method.

[0045] The present application provides a method for specifically enriching glycosylated peptide segments based on the above-mentioned Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA), which is as follows:

[0046] Take the above-mentioned Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA), and wash the magnetic beads with 80% ACN 1% TFA;

[0047] Dissolve the heat-dried peptide segments in 60-90% ACN 0.1-2% TFA, mix with the magnetic beads, and incubate at 30°C and 1500 rpm. After incubation, discard the supernatant by magnetic attraction;

[0048] Add 60-90% ACN 0.1-2% TFA to the magnetic beads and mix well, shake at 30°C and 300-2500 rpm for 5 min, and remove the supernatant by magnetic attraction, without repeating or repeating multiple times;

[0049] Add 5-50% ACN 0.1-2% TFA to the magnetic beads, shake at 4-60°C and 300-2500 rpm for 10 min, then remove the supernatant by magnetic attraction, repeat twice, and collect the glycopeptides, centrifuge at 4°C and 20000 xg for 10 min, take the supernatant and vacuum concentrate and dry at 45°C, to obtain the enriched glycosylated peptide segments;

[0050] Alternatively, dissolve the heat-dried peptide segments in 80% ACN 1% TFA, mix with the magnetic beads, and incubate at 30°C and 1500 rpm. After incubation, discard the supernatant by magnetic attraction;

[0051] Optionally, 80% ACN 1% TFA is added to the magnetic beads, and after shaking at 30 ℃ and 1500 rpm for 5 min, the supernatant is removed by magnetic attraction, and the operation is repeated or repeated multiple times.

[0052] Optionally, 20% ACN 1% TFA is added to the magnetic beads, and after shaking at 30 ℃ and 1500 rpm for 10 min, the supernatant is removed by magnetic attraction, and the operation is repeated twice, and the collected glycopeptides are collected.

[0053] In the present application, the peptide segment includes but is not limited to a hot-dried peptide segment, a freeze-dried peptide segment. The peptide segment sample without liquid introduction after salting out can be used in the present application.

[0054] The analyte added in the method for specifically enriching glycosylated peptides used in the present application can be a composition obtained from or derived from a subject, which contains cells and / or other molecular entities to be characterized and / or identified according to physical, biochemical, chemical and / or physiological characteristics. For example, the sample refers to any sample derived from a subject, which is expected or known to contain cells and / or molecular entities to be characterized. The sample includes but is not limited to one or a combination of a tissue sample (such as a tumor tissue sample), a primary or cultured cell or cell line, a cell supernatant, a cell lysate, a platelet, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract.

[0055] The present application provides an application of the aforementioned Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) in glycosylated peptide segment enrichment analysis, which selectively enriches glycosylated peptide segments by Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA).

[0056] The present application provides a method for glycosylated peptide segment proteomics analysis based on the aforementioned method for specifically enriching glycosylated peptide segments, which uses mobile phase A and mobile phase B, wherein mobile phase A uses A liquid, and the components are 100% water, 0.1% FA, and mobile phase B uses B liquid, and the components are 80% ACN, 0.1% FA, and the method comprises the following steps:

[0057] 1) Dissolve the enriched glycosylated peptide segments prepared by the method as described above with 10 μL of A liquid, and centrifuge at 14000 g at 4 ℃ for 20 min;

[0058] 2) Thermo Fisher Easy-nLC 1200 or U3000 liquid chromatography system is selected, and tandem separation is carried out by the following effective gradient: 0 min, 5% mobile phase B; 0-15 min, 5% mobile phase B; 15-25 min, 8% mobile phase B; 25-35 min, 10% mobile phase B; 35-45 min, 16% mobile phase B; 45-54 min, 32% mobile phase B; 54-64 min, 95% mobile phase B; 65 min, 6% mobile phase B, using a Q Exactive HF-X mass spectrometer and a Nanospray FlexTM (NSI) ion source, setting the ion spray voltage to 2.2 kV, the ion transfer tube temperature to 320 DEG C, the mass spectrometer to use a data-dependent acquisition mode, the mass spectrometer full scan range to be m / z 350-1800, the first mass spectrometer resolution to be 60 k, the AGC to be 3x10 6 , the C-trap maximum injection time to be 50 ms; the parent ions with the ion intensity TOP 15 in the full scan are selected to use high-energy collision fragmentation (HCD) method to break, and secondary mass spectrometry detection is carried out, the secondary mass spectrometer resolution is set to 45 k, the AGC is 2x10 5 , the maximum injection time is 120 ms, the peptide fragment collision energy is set to 34%, the mass spectrometry detection original data are generated, the mass spectrometry data are analyzed by Fragpipe software, the target database is UniProt Mouse, the search parameter is set to trypsin full enzyme cutting, 2 missed cutting sites are allowed, the parent ion mass error is set to 20 ppm, and the data are exported after the search is ended for analysis.

[0059] Further, 1 ug of the supernatant sample is injected for liquid chromatography-mass spectrometry detection after the centrifugation in step 1) is completed.

[0060] The application provides application of the preparation method in the equipment or system for automatically producing the glycosylated peptide magnetic nanoparticles.

[0061] The application has the following advantages and beneficial effects:

[0062] The application provides a glycosylated peptide enrichment method based on functionalized magnetic nanoparticles, the core of which is to enrich the peptides by specific binding of chemically modified magnetic nanoparticles and glycosylated peptides, thereby solving the problems of high cost, complicated operation and limited identification throughput, and providing an efficient, stable and easy-to-operate technical process for glycoproteomics research.

[0063] The key point of the present application is to develop a new method for synthesizing glycopeptide enrichment material, which realizes high selectivity and high efficiency of glycopeptide enrichment by introducing amphiphilic functional groups and optimizing the structure of the material. The specific protection points include: 1. Preparation method of functionalized magnetic nanoparticles: by chemical modification, allyl functional groups are introduced on the surface of Fe3O4@SiO2 magnetic nanoparticles (Fe3O4@SiO2@a-mSi), and further coated with poly(MBAAm-co-SBMA) zwitterionic polymer layer, forming a composite material with high hydrophilicity and specific binding capacity. 2. Efficient enrichment method of glycopeptide: the specific operation conditions of glycopeptide enrichment include the composition and the use volume of 80% ACN 1% TFA and 20% ACN 1% TFA. The incubation conditions (such as 30 ℃, 1500 rpm, 1 hour) and the optimization parameters of the washing step in the enrichment process. 3. Use of mass spectrometry and liquid chromatography parameters. 4. The operation efficiency of the material is improved. 5. Biocompatibility in complex samples: functionalized magnetic nanoparticles not only have high hydrophilicity and specific binding capacity, but also show excellent biocompatibility and stability, which are suitable for analysis of complex biological samples (such as serum, urine, tissue lysate). BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 is a schematic diagram of magnetic bead synthesis.

[0065] Figure 2 is a transmission electron micrograph of synthesized Fe3O4, Fe3O4@SiO2@a-mSi, Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) respectively.

[0066] Figure 3 is a comparison diagram of the existing commercial method and the method of the present application for glycopeptide enrichment analysis process.

[0067] Figure 4 is the off-machine map data, wherein (a)-(b): the present application, (c)-(d): ZIC-HILIC.

[0068] Figure 5 is a comparison of the repeatability of the present application and the commercial repeatability, wherein (a), (c): N-linked glycosylation, (b), (d): O-linked glycosylation. DETAILED DESCRIPTION

[0069] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application. The experimental methods used in the following embodiments are conventional methods, unless otherwise specified. The materials and reagents used in the following embodiments are commercially available, unless otherwise specified.

[0070] Embodiments

[0071] 1. Experimental methods

[0072] 1.1 Synthesis of Fe3O4 magnetic nanoparticles

[0073] Dissolve 10.4 g FeCl3·6H2O and 4.0 g FeCl2·4H2O in 100 mL deionized water, and degas with nitrogen for 15 minutes. Heat the solution to 80 ℃, and then slowly drop 15 mL NH4OH (32%) solution. Keep the reaction for 15 minutes, and then separate the solid with a magnet. Finally, wash three times with 0.1 mol / L NaCl solution to remove unreacted ions and byproducts, and dry the Fe3O4 nanoparticles in a 50 ℃ oven.

[0074] 1.2 Preparation of Fe3O4@SiO2 core-shell structure

[0075] Disperse 1 g of synthesized Fe3O4 nanoparticles in a mixed solution of 200 mL distilled water, 50 mL ethanol and 10 mL concentrated ammonia (32%). Slowly drop 0.5 g of tetraethyl orthosilicate (TEOS) under vigorous stirring, and continue to stir for 6 hours to make SiO2 uniformly coated on the surface of Fe3O4. After the reaction is completed, separate the silica-coated Fe3O4 nanoparticles with a magnet, and wash three times with ethanol and deionized water alternately to remove unreacted TEOS and byproducts.

[0076] 1.3 Synthesis of allyl-functionalized Fe3O4@SiO2 (Fe3O4@SiO2@a-mSi)

[0077] The above reaction completed Fe3O4@SiO2nanoparticles were dispersed in 200 mL distilled water, 50 mL ethanol and 10 mL concentrated ammonia solution, and 3 g CTAB was added. Under vigorous stirring, 23.5 mL TEOS and 5 g allyltrimethoxysilane (ATMS) were slowly added dropwise, and the reaction was carried out for 6 hours. After the reaction was completed, the allyl-functionalized mesoporous SiO2coated magnetic particles (Fe3O4@SiO2@a-mSi) were separated by a magnet. Finally, the nanoparticles were washed with ethanol and water alternately three times to remove CTAB and unreacted monomers, and were dried in an oven at 50 °C.

[0078] 1.4 Synthesis of poly(MBAAm-co-SBMA) coating layer

[0079] Fe3O4@SiO2@a-mSi nanoparticles, 0.2 g of sulfobetaine methacrylate (SBMA) and 1.0 g of N,N'-methylenebisacrylamide (MBAAm) were dispersed in 100 mL ACN solution. The mixed solution was transferred to a 250 mL single-necked round-bottom flask, and purged with nitrogen for 30 minutes to remove dissolved oxygen. Then, 0.0225 g of azobisisobutyronitrile (AIBN) was added as an initiator, and the temperature was raised to 75 °C for free radical polymerization. The reaction system was connected with a Dean-Stark receiver, and heated to 75 °C for 10 minutes. Then the oil bath temperature was slowly raised to 100 °C, and kept at reflux for 1 hour, and about 35 mL of ACN was distilled off. After the reaction was completed, it was cooled to room temperature and stirred for 1 hour. Finally, the nanoparticles were washed with ACN three times to remove unreacted monomers and oligomers, and dried at 50 °C for 12 hours.

[0080] 1.5 Protein extraction and desalination

[0081] Mouse brain tissue was cut into small pieces and placed in a grinding tube with 2 large steel beads. After quick freezing in liquid nitrogen, the tissue was ground into powder using a grinder. 5% sodium dodecyl sulfate (SDS) lysis buffer was added to the grinding tube, and after homogenization, non-contact ultrasonic treatment was used for 15 minutes, followed by centrifugation at 14,000 g for 10 minutes at 4°C. The supernatant was collected and the protein was quantified using the BCA method. Dithiothreitol (DTT) was added to the supernatant to a final concentration of 5 mM, and heated at 37°C for 30 minutes. After cooling to room temperature, iodoacetamide (IAA) was added to a final concentration of 15 mM, and reacted in the dark for 30 minutes. Diluted 5 times with 25 mM NH4HCO3, followed by the addition of trypsin (enzyme to substrate ratio of 1:50), and enzymolysis at 37°C overnight. An equal volume of 1% formic acid (FA) was added to the enzymolysis solution, vortexed for 5 minutes, and then centrifuged at 16,000 g for 15 minutes at 4°C. The supernatant was collected. The sample was desalted using a C18 solid phase extraction column, and the flow-through was collected and vacuum concentrated and dried.

[0082] 1.6 Glycopeptide enrichment and proteomic analysis

[0083] 10 mg of functionalized magnetic nanoparticles were weighed, and 400 μL of 80% ACN 1% TFA was used to wash the magnetic beads 3 times. 200 μg of heat-dried peptides were dissolved in 300 μL of 80% ACN 1% TFA, mixed with the magnetic beads, and incubated on a shaker at 30°C and 1500 rpm for 1 h. After incubation, the supernatant was removed by magnetic attraction. 400 μL of 80% ACN 1% TFA was added to the magnetic beads, mixed well by blowing, and shaken on a shaker at 30°C and 1500 rpm for 5 min. The supernatant was removed by magnetic attraction. This washing step was repeated 3 times. 250 μL of 20% ACN 1% TFA was added to the magnetic beads, shaken on a shaker at 30°C and 1500 rpm for 10 min, and the supernatant was removed by magnetic attraction and transferred to a new tube. This elution step was repeated twice, and a total of 500 μL of glycopeptides were collected. Centrifugation at 20,000 x g for 10 min at 4°C, transfer of the supernatant to a new tube, and vacuum concentration and drying at 45°C.

[0084] Prepare mobile phase A liquid (100% water, 0.1% FA) and B liquid (80% ACN, 0.1% FA). Use 10 μL of A liquid to dissolve the freeze-dried powder, centrifuge at 4 ℃ for 20 min at 14000 g, and inject 1 μg of supernatant sample for liquid chromatography-mass spectrometry detection. Select ThermoFisher Easy-nLC 1200 or U3000 liquid chromatography system for tandem separation by the following effective gradient: 0 min, 5% mobile phase B; 0-15 min, 5% mobile phase B; 15-25 min, 8% mobile phase B; 25-35 min, 10% mobile phase B; 35-45 min, 16% mobile phase B; 45-54 min, 32% mobile phase B; 54-64 min, 95% mobile phase B; 65 min, 6% mobile phase B. Use Q Exactive HF-X mass spectrometer and Nanospray Flex™ (NSI) ion source, set the ion spray voltage to 2.2 kV, the ion transfer tube temperature to 320 ℃, the mass spectrometer to data-dependent acquisition mode, the mass spectrometry full scan range to m / z 350-1800, the first mass spectrometry resolution to 60 k, the AGC to 3×10 6 , and the C-trap maximum injection time to 50 ms; select the TOP 15 parent ions in the full scan for fragmentation by high-energy collision-induced dissociation (HCD) method, perform secondary mass spectrometry detection, set the secondary mass spectrometry resolution to 45 k, the AGC to 2×10 5 , the maximum injection time to 120 ms, the peptide fragmentation collision energy to 34%, and generate mass spectrometry detection raw data (.raw). Perform database searching analysis on the mass spectrometry data by Fragpipe software, set the target database to UniProt Mouse, the search parameters to trypsin full enzyme digestion, allow 2 missed cleavage sites, and the parent ion mass error to 20 ppm. Export the data for analysis after the database searching is completed.

[0085] 2. Experimental results

[0086] Prepare a new type of magnetic nanomaterial with MBAAm and SBMA on the surface and use it for glycosylated peptide enrichment and proteomics analysis, according to the synthetic route shown in Figure 1 Prepare the magnetic nanomaterial with SBMA on the surface of the magnetic nanomaterial.

[0087] The transmission electron microscopy images of the synthesized Fe3O4 magnetic beads, Fe3O4@SiO2@a-mSi magnetic beads, and Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) magnetic beads are shown in Figure 2 The surface material coating of the magnetic beads is confirmed to be successful.

[0088] The experimental procedure for glycosylation enrichment is as followsFigure 3 As shown, compared with the traditional commercial ZIC-HILIC material, the application does not need sample loading process, and can greatly save time in washing and elution process, thus greatly improving the efficiency.

[0089] The mass spectrometry was used to detect the ability of the application to enrich glycopeptides, and the offline data were as shown in Figure 4 The software fragpipe (v22) was selected for database searching, and the data statistics were as shown in Table 1. The Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) material could identify an average of 2733 N-linked glycopeptides and 1777 O-linked glycopeptides.

[0090] Table 1. Statistics table of the number of glycopeptides identified by mass spectrometry (application vs. prior art)

[0091]

[0092] In addition, the material stability data were as shown in Figure 5 The experimental repeatability of the material of the application in glycopeptide enrichment was equivalent to that of the market mainstream commercial material, and there were more than 1400 intersection peptides, which fully embodied the stable performance of the material in identifying glycopeptides in complex biological samples.

Claims

1. A method for preparing magnetic nanoparticles that specifically separate glycosylated peptides, the method comprising: 1) Fe3O4@SiO2 nanoparticles were dispersed in a concentrated ammonia ethanol solution, CTAB was added, and TEOS and allyltrimethoxysilane were slowly added dropwise under vigorous stirring. After the reaction was completed, the allyl-functionalized mesoporous SiO2-coated magnetic particles were separated by a magnet. The particles were washed alternately with ethanol and water to remove CTAB and unreacted monomers, and then dried in an oven at 50 °C to obtain Fe3O4@SiO2@a-mSi. 2) The dried allyl-functionalized mesoporous SiO2-coated magnetic particles, SBMA, and MBAAm were added to an ACN solution to remove dissolved oxygen. AIBN was then added, and the mixture was heated to 75 °C for free radical polymerization. 5-60% of the volume of ACN was distilled off by slowly increasing the temperature from 75 °C to 100 °C for 10 min. The mixture was cooled to room temperature and stirred. The nanoparticles were washed with ACN to remove unreacted monomers and oligomers, and then dried at 50 °C to obtain the final Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA). The ethanol-ammonia solution consists of distilled water, ethanol, and concentrated ammonia solution in a volume ratio of 15~23:3~8:0.5~2. The ratio of Fe3O4@SiO2 nanoparticles to concentrated ammonia ethanol solution, CTAB, TEOS, and allyltrimethoxysilane is 0.5~2 g: 240~300 mL: 0.5~8 g: 5~40 mL: 1~20 g; In step 2), the ratio of allyl-functionalized mesoporous SiO2-coated magnetic particles, SBMA, MBAAm, and ACN solution is 0.01~1 g: 0.1~100 g: 0.1~100 g: 20~500 mL. The AIBN is used as an initiator, and the ratio of AIBN to ACN solution is 0.01~5 g: 20~500 mL.

2. According to the preparation method of claim 1, 35% volumetric ACN is distilled off.

3. According to the preparation method of claim 1, the components of the ethanol-ammonia solution are distilled water, ethanol and concentrated ammonia solution, with a volume ratio of 20:5:

1.

4. According to the preparation method of claim 1, the ratio of the Fe3O4@SiO2 nanoparticles to concentrated ammonia ethanol solution, CTAB, TEOS, and allyltrimethoxysilane is 1 g: 260 mL: 3 g: 23.5 mL: 5 g.

5. According to the preparation method of claim 1, in step 2), the ratio of allyl-functionalized mesoporous SiO2 coated magnetic particles, SBMA, MBAAm and ACN solution is 40 mg: 0.2 g: 1 g: 100 mL.

6. The preparation method according to claim 1, wherein the AIBN is used as an initiator and the ratio of AIBN to ACN solution is 0.0225 g: 100 mL.

7. The preparation method according to claim 1, wherein the Fe3O4@SiO2 nanoparticles are synthesized by the following method: Fe3O4 nanoparticles were dispersed in a mixed solution of distilled water, ethanol, and 32% concentrated ammonia. TEOS was slowly added dropwise under vigorous stirring, and stirring was continued to make SiO2 uniformly coat the Fe3O4 surface. After the reaction was completed, the silica-coated Fe3O4 nanoparticles were separated by a magnet and washed alternately with ethanol and deionized water to remove unreacted TEOS and byproducts.

8. In the preparation method according to claim 7, the mixing ratio of distilled water, ethanol, and 32% concentrated ammonia is 1~100:5:0.1~5.

9. The preparation method according to claim 7, wherein the mixing ratio of distilled water, ethanol, and 32% concentrated ammonia is 20:5:

1.

10. The preparation method according to claim 7, wherein the ratio of Fe3O4 nanoparticles to the mixed solution is 0.2~2 g: 100~500 mL.

11. The preparation method according to claim 7, wherein the ratio of Fe3O4 nanoparticles to the mixed solution is 1 g: 260 mL.

12. The preparation method according to claim 7, wherein the ratio of Fe3O4 nanoparticles to TEOS is 0.3~3 g: 0.3~0.7 g.

13. The preparation method according to claim 7, wherein the ratio of Fe3O4 nanoparticles to TEOS is 1 g: 0.5 g.

14. The preparation method according to claim 7, wherein the Fe3O4 nanoparticles are synthesized by the following method: FeCl3·6H2O and FeCl2·4H2O were dissolved in deionized water and degassed with nitrogen. The solution was heated to 80 °C, and then 32% NH4OH solution was slowly added dropwise while maintaining the reaction. The solid was separated by a magnet. Finally, the solid was washed with 0.1 mol / L NaCl solution to remove unreacted ions and byproducts, and the Fe3O4 nanoparticles were dried in an oven at 50 °C.

15. The preparation method according to claim 14, wherein the ratio of deionized water to FeCl3·6H2O, FeCl2·4H2O, and 32% NH4OH solution is 50~300 mL: 1~30 g: 1~30 g: 8~20 mL.

16. According to the preparation method of claim 14, the ratio of deionized water to FeCl3·6H2O, FeCl2·4H2O, and 32% NH4OH solution is 100 mL: 10.4 g: 4 g: 15 mL.

17. Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) prepared by any one of claims 1-16.

18. A method for specifically enriching glycosylated peptides based on Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) as described in claim 17, wherein the method is as follows: Take the Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) as described in claim 17 and clean the magnetic beads with 80% ACN and 1% TFA; Dissolve the heat-dried peptides thoroughly in 60-90% ACN and 0.1-2% TFA, then mix with magnetic beads and incubate at 4-60 °C and 300-2500 rpm. After incubation, magnetically remove the supernatant. Add 60-90% ACN and 0.1-2% TFA to the magnetic beads and mix well. Shake at 4-60 ℃ and 300-2500 rpm for 5 minutes. Remove the supernatant by magnetic attraction. Do not repeat or repeat multiple times. Add 5-50% ACN and 0.1-2% TFA to magnetic beads, shake at 4-60 ℃ and 300-2500 rpm for 10 min, magnetically aspirate the supernatant, repeat twice, collect the glycopeptides, centrifuge at 20000 xg at 4 ℃ for 10 min, take the supernatant and vacuum concentrate and dry at 45 ℃ to obtain enriched glycosylated peptides.

19. The method according to claim 18, wherein the heat-dried peptide is fully dissolved in 80% ACN and 1% TFA, then mixed with magnetic beads, and incubated at 30 °C and 1500 rpm, and the supernatant is magnetically removed after incubation.

20. The method according to claim 18, wherein 80% ACN and 1% TFA are added to the magnetic beads and mixed well, shaken at 30 °C and 1500 rpm for 5 min, and the supernatant is removed by magnetic adsorption, without repeating or repeating multiple times.

21. According to the method of claim 18, 20% ACN and 1% TFA are added to the magnetic beads, and the mixture is shaken at 30 °C and 1500 rpm for 10 min. The supernatant is then magnetically aspirated, and the process is repeated twice to collect the glycopeptides.

22. An application of Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA) as described in claim 17 in the enrichment analysis of glycosylated peptides, wherein the application selectively enriches glycosylated peptides using Fe3O4@SiO2@a-mSi@poly(MBAAm-co-SBMA).

23. A method for proteomics analysis of glycosylated peptides based on any one of claims 18-21, the method using mobile phase A and mobile phase B, wherein mobile phase A uses solution A, composed of 100% water and 0.1% FA, and mobile phase B uses solution B, composed of 80% ACN and 0.1% FA, the method comprising the following steps: 1) Dissolve the enriched glycosylated peptides prepared by the method described in claim 7 in 10 μL of solution A, and centrifuge at 14000 g for 20 min at 4 °C; 2) A Thermo Fisher Easy-nLC 1200 or U3000 liquid chromatography system was used for tandem separation using the following effective gradients: 0 min, 5% mobile phase B; 0-15 min, 5% mobile phase B; 15-25 min, 8% mobile phase B; 25-35 min, 10% mobile phase B; 35-45 min, 16% mobile phase B; 45-54 min, 32% mobile phase B; 54-64 min, 95% mobile phase B; 65 min, 6% mobile phase B. A Q Exactive HF-X mass spectrometer and a Nanospray Flex™ (NSI) ion source were used. The ion spray voltage was set to 2.2 kV, the ion transfer tube temperature to 320 °C, and the mass spectrometry was performed in data-dependent acquisition mode. The full scan range was m / z 350-1800, the first-order mass spectrum resolution was set to 60 k, and the AGC was 3 × 10⁻⁶. 6 The maximum C-trap injection time was 50 ms. The top 15 precursor ions by ion intensity in the full scan were fragmented using high-energy collisional fragmentation (HCD) and detected by secondary mass spectrometry. The resolution of the secondary mass spectrometry was set to 45 k, and the AGC was 2 × 10⁻⁶. 5 The maximum injection time was 120 ms, the peptide fragmentation collision energy was set to 34%, and raw mass spectrometry data were generated. The mass spectrometry data were analyzed by searching the database using Fragpipe software. The target database was UniProt Mouse, and the search parameters were set to full digestion of trypsin, allowing 2 missed cleavage sites, and the precursor ion mass error was set to 20 ppm. After the database search was completed, the data was exported for analysis.

24. According to the method of claim 23, after centrifugation in step 1), 1 μg of the supernatant is taken and injected for liquid chromatography-mass spectrometry (LC-MS) analysis.

25. The application of the preparation method according to any one of claims 1-16 in an equipment or system for the automated production of magnetic nanoparticles for the specific separation of glycosylated peptides.

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