CN110665465A - Magnetic covalent organic framework material for glycopeptide enrichment and preparation method and application thereof - Google Patents
Magnetic covalent organic framework material for glycopeptide enrichment and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a magnetic covalent organic framework material for glycopeptide enrichment, and a preparation method and application thereof3O4Nano magnetic ball coated on Fe3O4The preparation method comprises the following steps: preparation of Fe3O4/COFs nanoparticles, preparation of Fe3O4a/COF-GSH material; the magnetic covalent organic framework material is shown to be enriched in glycopeptideThe method has the advantages of high selectivity, strong binding capacity, high enrichment efficiency, good recovery efficiency and the like, has very important significance in the research of the glycosylation process of physiological behaviors, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of biological materials, and relates to a magnetic covalent organic framework material, a preparation method thereof and application of the magnetic covalent organic framework material in enrichment of endogenous glycopeptides.
Background
The existing magnetic covalent organic framework material is composed of magnetic nanospheres and a hydrophilic Covalent Organic Framework (COFs) wrapped on the surfaces of the magnetic nanospheres, has good magnetic response performance, and has the unique properties of high porosity, highly ordered mesopores, abundant organic ligands, stable structure and the like of the COFs. Therefore, in recent years, magnetic covalent organic framework materials have attracted much attention and have been widely used in biomedical fields, especially in protein or polypeptide separation, drug delivery, magnetic resonance imaging, and the like.
At present, the enrichment application of the magnetic covalent organic framework material to glycopeptide basically adopts a hydrophilic interaction chromatography method, namely, the glycopeptide is enriched only by utilizing the hydrophilicity of the covalent organic framework. Xiangmin Zhang et al discloses a preparation method of a magnetic graphene composite material modified by a covalent organic framework, and provides an application of the composite material in glycopeptide enrichment, the magnetic graphene composite material modified by the covalent organic framework uses graphene as a substrate material, then nano magnetic spheres are modified on the surface of the graphene, finally a layer of covalent organic framework layer using 2,3,6,7,10, 11-hexahydroxy triphenyl (HHTP) and terephthalic diboronic acid (BDBA) as organic ligands is modified on the surface of the graphene modified by the magnetic spheres, so as to obtain the magnetic graphene composite material modified by the covalent organic framework, the magnetic composite material is used for glycopeptide enrichment (Self-assembling organic molecular functional doped graphene inorganic graphene nano material used for N-linked graphene oxide enrichment, 20150, 1075, xiangmin Zhang, etc.).
However, due to the limitation of hydrophilic properties of covalent organic framework materials and the lack of flexible functional modification, the above-mentioned covalent organic framework based composite materials are not ideal for glycopeptide enrichment.
Disclosure of Invention
The present invention aims to solve the above problems in the prior art, and provide a magnetic covalent organic framework material for glycopeptide enrichment and a preparation method thereof, so as to improve the hydrophilicity of the covalent organic framework material and achieve high efficiency enrichment of glycopeptide.
The method is used for enriching glycopeptidesMagnetic covalent organic framework material of (1), from Fe3O4Nano magnetic ball coated on Fe3O4A Covalent Organic Framework (COF) layer on the surface of the nano magnetic sphere and Glutathione (GSH) grafted on the covalent organic framework layer; the covalent organic framework layer is prepared by mixing 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde according to the mass ratio of 1:1.5 a cellular structure obtained by an addition reaction, said glutathione being grafted onto vinyl groups of the covalent organic framework layer.
The magnetic covalent organic framework material for glycopeptide enrichment is complete and spherical, has uniform and narrow particle size distribution, has an average particle size of about 240-300 nm, and is suitable for enrichment and separation of protein and polypeptide. The magnetic covalent organic framework material is superparamagnetic ferroferric oxide (Fe)3O4Nano magnetic ball) as an inner core, has high magnetic saturation strength, and thus has good magnetic response performance to an external magnetic field; fe used in the invention3O4The nano magnetic ball accounts for about 48 percent of the mass of the magnetic covalent organic framework material, so that the saturation magnetization of the magnetic covalent organic framework material reaches 45emu g-1Left and right. Coated with Fe3O4The COF layer on the surface of the nano magnetic sphere has high porosity, highly ordered mesopores and large specific surface area, thereby being beneficial to enrichment application of glycopeptide. The COF layer is prepared from two organic ligands of 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde. Wherein, for the 2, 5-divinyl-1, 4-benzene dicarbaldehyde organic ligand, on one hand, the ligand is one of components for synthesizing a covalent organic framework, and the remaining carbon-carbon double bond of the ligand can be used for further introducing zwitter-ion glutathione with high hydrophilic performance as a reaction active site. In the invention, Glutathione (GSH) is modified on a COF layer by utilizing a high-efficiency mercaptoalkene clicking method, and the GSH is zwitter ions, so that the hydrophilic performance of the magnetic covalent organic framework material based on COFs is improved to a great extent, the limitation of the hydrophilic performance of the existing COFs composite material is overcome, and the GSH can realize high-efficiency enrichment of glycopeptides.
The preparation method of the magnetic covalent organic framework material for glycopeptide enrichment is mainly realized through an epitaxial growth mechanism, a covalent organic framework layer (two ligands form a COF layer through covalent bond action) is wrapped on the surface of a magnetic ball through an epitaxial growth method under a mild condition, and then high-hydrophilicity zwitterionic glutathione is modified on the surface of the covalent organic framework layer through a high-efficiency mercaptoalkene clicking method.
The invention discloses a preparation method of a magnetic covalent organic framework material for glycopeptide enrichment, which comprises the following steps:
(1) preparation of Fe3O4/COFs nanoparticles
Under the ultrasonic condition, Fe3O4Mixing and dispersing nanometer magnetic spheres, 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde in dimethyl sulfoxide uniformly to form a mixed solution; then, under the ultrasonic condition, dropwise adding acetic acid into the mixed solution to form a reaction system; then standing and incubating the obtained reaction system at room temperature for 10-30 minutes; after the incubation is finished, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, washing the obtained solid product to remove unreacted materials adsorbed on the surface of the solid product, and obtaining covalent organic framework layer coated Fe3O4Nanoparticles of nano-magnetic spheres, abbreviated as Fe3O4/COFs nanoparticles; said Fe3O4The mass ratio of the nano magnetic ball to the 1,3, 5-tri (4-aminophenyl) benzene is 1: (0.2-1), the mass ratio of the 1,3, 5-tri (4-aminophenyl) benzene to the 2, 5-divinyl-1, 4-benzene dicarbaldehyde is 1:1.5, and the volume ratio of the mass sum of the two ligands of the 1,3, 5-tri (4-aminophenyl) benzene and the 2, 5-divinyl-1, 4-benzene dicarbaldehyde to the acetic acid is (30-210): 1, the unit of mass is mg, and the unit of volume is mL;
(2) preparation of Fe3O4Magnetic covalent organic framework material of/COF-GSH
Azodiisobutyronitrile, glutathione and Fe3O4the/COFs nano particles are sequentially added into a composite solvent to form a mixed solution, and then under the protection of nitrogen, mercaptoalkene click is carried out on the obtained mixed solution at the temperature of 60-80 DEG CReaction to Fe3O4The surface of the COFs nanoparticle is yellow; after finishing mercaptoene click reaction, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, washing the obtained solid product to remove unreacted materials adsorbed on the surface of the solid product, and drying to obtain the magnetic covalent organic framework material for glycopeptide enrichment, Fe for short3O4a/COF-GSH material; the composite solvent is obtained by uniformly mixing ethanol and deionized water according to the volume ratio of 1:3, and Fe is contained in the mixed solution3O4The mass ratio of the/COFs nano particles to the glutathione is 1 (0.3-1); the mass ratio of the glutathione to the azodiisobutyronitrile is 1 (0.05-0.25).
The preparation method of the magnetic covalent organic framework material for glycopeptide enrichment comprises the step (1) of Fe3O4The nano magnetic ball is mainly prepared by taking ferric chloride, sodium acetate and sodium citrate as raw materials and using ethylene glycol as a solvent to synthesize superparamagnetic ferroferric oxide nanospheres with the particle size of 200-250 nm by a hydrothermal method; in addition, the particle size distribution of the magnetic spheres can be regulated and controlled by regulating the hydrothermal reaction time. Preparation of Fe3O4Specific implementations of nano-magnetic spheres can be obtained by conventional preparation methods disclosed in The prior art, see The design and synthesis of a hydrophilic core-shell structured magnetic metal-organic frame as a novel immobilized magnetic metal-inorganic plate for phosphor layer research chemistry, 2014,50,6228, Chunhui Deng et al, and Ti4+Immobilized multilayered polysaccharide coated magnetic nanoparticles for high selectivity evaluation of phosphopeptides J. mater. chem. B2014, 2,4473-4480, Hanfa Zou et al.
In the preparation method of the magnetic covalent organic framework material for glycopeptide enrichment, in the step (1), two ligands are prepared into a COF layer through an addition reaction, wherein acetic acid is used as a catalyst, and Fe is used as a catalyst3O4The nano magnetic ball is used as a carrier. Under the ultrasonic condition, Fe3O4Mixing and uniformly dispersing the nano magnetic spheres, 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde by ultrasonic waves for 5-10 min generally; dimethyl sulfoxideThe sulfone is used in such an amount that it completely dissolves 1,3, 5-tris (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzenedicarboxaldehyde and allows Fe to be contained3O4The nano magnetic spheres are uniformly dispersed. And then, dropwise adding acetic acid into the mixed solution under the ultrasonic condition, wherein the dropwise adding time is generally controlled to be 5-15 min. After the dropwise addition of the acetic acid is finished, the obtained mixed solution is kept stand and incubated for 10-30 minutes at room temperature, and the solid product in the obtained reaction solution is Fe3O4/COFs nanoparticles. So that the reaction liquid is subjected to solid-liquid separation and the solid product is washed to obtain Fe3O4/COFs nanoparticles. The purpose of washing is to remove unreacted materials adsorbed on the surface of the solid product, and the washing mode adopted in the invention is as follows: and washing the separated solid product with anhydrous tetrahydrofuran, anhydrous methanol, ethanol and deionized water in sequence, wherein each washing solution is generally washed for 3-5 times.
In the preparation method of the magnetic covalent organic framework material for glycopeptide enrichment, in the step (2), the zwitterionic glutathione is modified on the surface of the covalent organic framework layer by a high-efficiency mercaptoalkene clicking method. The reaction needs to be carried out under the condition of no oxygen, and before the mixed liquid obtained by the invention is reacted under the protection of nitrogen, oxygen removal treatment is carried out to remove oxygen in the mixed liquid and above the mixed liquid in the reactor. The specific treatment method comprises the following steps: firstly, azodiisobutyronitrile, glutathione and Fe3O4Adding the/COFs nano particles into a composite solvent containing deionized water and ethanol in sequence, and ultrasonically mixing uniformly; then, deoxidizing the mixed solution in a nitrogen purging mode, wherein the deoxidizing time is about 0.5-2 h; and removing oxygen in the reactor by adopting a vacuum-pumping-nitrogen-introducing circulating operation mode, generally circulating for 3-5 times, firstly vacuumizing the reactor until the vacuum degree is not more than 100Pa in each circulation, and then introducing nitrogen to the normal pressure. For facilitating the later cleaning operation, the cleaning agent is used for azodiisobutyronitrile, glutathione and Fe3O4The COFs nano particles are sequentially added into a composite solvent, a container for ultrasonic mixing and a reactor for subsequent mercaptoene click reaction can be different, after the mixed solution and the reactor are respectively deoxygenated, the deoxygenated mixed solution is transferred into the deoxygenated reactor,and then reacting at 60-80 ℃ under the condition of magnetic stirring until the surfaces of the nanoparticles are yellow, wherein the magnetic nanoparticles in the mixed solution are well dispersed and do not adhere to the wall, and the reaction time is about 6-24 hours generally. The solid product in the obtained reaction solution is Fe3O4a/COF-GSH material. So that the reaction solution is subjected to solid-liquid separation, and the solid product is washed and dried to obtain Fe3O4a/COF-GSH magnetic covalent organic framework material. The purpose of washing is to remove unreacted materials adsorbed on the surface of the solid product, and the washing mode adopted in the invention is as follows: and washing the separated solid product with ethanol and deionized water in sequence, wherein each washing solution is generally washed for 3-5 times.
The invention further provides application of the magnetic covalent organic framework material in enrichment of endogenous glycopeptides. The magnetic covalent organic framework material has a good enrichment effect on glycopeptides in a biological sample, particularly shows extremely excellent performance in the process of enriching endogenous glycopeptides in human saliva, and has a very important significance in the process of researching the glycosylation of physiological behavior proteins.
The operation of enriching glycopeptide by using the magnetic covalent organic framework material is as follows:
(1) adsorption of glycopeptides: firstly, digesting glycosylated protein immunoglobulin G into glycopeptide by trypsin, diluting by using a buffer solution, then adding the magnetic covalent organic framework material of the invention, mixing and stirring uniformly, then shaking by using a shaking table for 10-60 min at room temperature to enrich the glycopeptide on the surface of the magnetic covalent organic framework material, and separating the magnetic covalent organic framework material with the glycopeptide adsorbed on the surface from the solution by using magnetic separation under the action of an external magnetic field;
(2) desorption of glycopeptides: and adding the magnetic covalent organic framework material with the glycopeptide adsorbed on the surface into desorption liquid to desorb the glycopeptide from the magnetic covalent organic framework material.
In the adsorption process of the glycopeptide, the glycopeptide is enriched by the modified high-hydrophilicity molecule GSH on the covalent organic framework by utilizing the principle of hydrophilic interaction chromatography. In the magnetic covalent organic framework material provided by the invention, the COF layer is a highly ordered mesoporous material with the aperture of about 3.6nm, and macromolecules (protein, exosome and the like) can be blocked outside the material, so that the enrichment application of the peptide segment of the endogenous glycopeptide is facilitated. The adsorbed glycopeptide is separated from the surface of the magnetic covalent organic framework material by changing the components of the buffer solution, so that the capture and separation of the glycopeptide are realized.
Compared with the prior art, the invention has the following beneficial effects:
1. the magnetic covalent organic framework material provided by the invention uses Fe3O4The nano magnetic ball is used as the inner core and is in Fe3O4A covalent organic framework composed of 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde is introduced to the surface of the nano magnetic sphere, residual carbon-carbon double bonds on the 2, 5-divinyl-1, 4-benzene dicarbaldehyde are taken as reaction sites, hydrophilic zwitter-ion GSH is modified through mercaptoene click reaction, the material not only shows good magnetic responsiveness, but also improves the hydrophilic performance of the magnetic covalent organic framework material based on COF to a great extent through the design of combining a COF layer with zwitter-ions, and has the advantages of high selectivity, strong binding capacity, high enrichment efficiency, good recovery efficiency and the like in the aspect of glycopeptide enrichment.
2. The magnetic covalent organic framework material provided by the invention is prepared from Fe3O4The covalent organic framework is introduced to the surface of the nano magnetic sphere, and has a high specific surface area and a highly ordered mesoporous structure, so that the magnetic covalent organic framework material is very suitable for enriching and separating protein or peptide fragments, especially endogenous glycopeptides.
3. The preparation method of the magnetic covalent organic framework material provided by the invention firstly adopts an epitaxial growth mode to carry out Fe3O4The COF layer is wrapped on the surface of the nano magnetic ball, then hydrophilic molecules GSH are grafted on the COF layer by adopting high-efficiency mercaptoalkene click reaction, the whole process is simple to operate, the reaction condition is mild, and the magnetic covalent organic framework material can be prepared in a short timeTherefore, the material is easy to be popularized in the field of biological medicine.
Drawings
Fig. 1 is a flow chart of a process for preparing a magnetic covalent organic framework material for glycopeptide enrichment and a flow chart for enriching glycopeptides, wherein a is a flow chart of a process for preparing a magnetic covalent organic framework material, and B is a flow chart of enriching glycopeptides by using a magnetic covalent organic framework material.
FIG. 2 is a graph showing the morphology of the magnetic nanomaterial prepared in example 1, where A is Fe3O4Scanning Electron Microscope (SEM) image of nano-magnetic spheres, B is Fe prepared in example 23O4Scanning Electron Microscopy (SEM) image of/COFs nanoparticles, C is Fe prepared in example 123O/Scanning Electron Microscope (SEM) image of COF-GSH material, D is Fe prepared in example 13O4Transmission Electron Microscopy (TEM) image of the nanomagnetic sphere, E being Fe prepared in example 23O4Transmission Electron Microscopy (TEM) image of/COFs nanoparticles, F being Fe prepared in example 123O4Scanning Electron Microscopy (SEM) image of/COF-GSH material.
FIG. 3 is an X-ray diffraction pattern of the magnetic nanomaterial prepared in example of the present invention, wherein a corresponds to Fe prepared in example 13O4Nanomagnetic spheres, b Fe prepared in example 23O4CoFs nanoparticles, c Fe prepared in example 123O4a/COF-GSH material.
FIG. 4 shows Fe prepared in example 12 of the present invention3O4Elemental composition characterization plot of/COF-GSH material.
FIG. 5 is a nitrogen adsorption-desorption curve and a mesoporous size distribution diagram of magnetic nanomaterials prepared in examples 2 and 12 of the present invention, wherein A is Fe prepared in example 23O4Nitrogen adsorption-desorption curves for/COFs nanoparticles, B being Fe prepared in example 123O4Nitrogen adsorption-desorption curve of/COF-GSH material, C being Fe prepared in example 23O4Mesoporous size distribution of/COFs nanoparticles, D being Fe prepared in example 123O4Mesoporous ruler of/COF-GSH materialCun distribution diagram.
FIG. 6 is a graph of the hysteresis loop of the magnetic nanomaterial prepared in the example of the present invention in the range of-18000 Oe to 18000Oe, wherein a corresponds to Fe prepared in example 13O4Nanomagnetic spheres, b Fe prepared in example 23O4CoFs nanoparticles, c Fe prepared in example 123O4a/COF-GSH material.
FIG. 7 is an MS spectrum of IgG digest in example 1 of the present invention, wherein A is the MS spectrum of IgG digest without enrichment and B is Fe prepared in example 123O4MS profile of the/COF-GSH material enriched treated immunoglobulin G digest.
Detailed Description
The technical solutions of the present invention will be described in detail and fully with reference to the accompanying drawings, which are used for describing the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The structure of the magnetic covalent organic framework material for glycopeptide enrichment provided by the invention is shown in figure 1, and the magnetic covalent organic framework material is made of Fe3O4Nano magnetic balls coated on Fe3O4A COF middle layer on the surface of the nano magnetic ball and zwitter ion GSH grafted on the COF layer. The invention prepares the magnetic covalent organic framework material based on the process flow given in figure 1. As shown in figure 1A, firstly, the hydrothermal method is utilized to prepare Fe3O4A nano magnetic ball; then according to epitaxial growth method to obtain Fe3O4Coating a COF (chip on film) intermediate layer on the surface of the nano magnetic ball to obtain a COF layer coated with Fe3O4Magnetic nanoparticles (Fe)3O4/COFs nanoparticles), and finally grafting zwitter-ion GSH to Fe through mercaptoene click reaction3O4Fe prepared on the surface of/COFs nano-particle3O4a/COF-GSH material.
Theoretically, the COF layer having a honeycomb structure with a six-membered ring as a basic unit as shown in fig. 1A was obtained by an addition reaction of two organic ligands of 1,3, 5-tris (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzenedicarboxaldehyde in a ratio of the amounts of the substances of 1: 1.5. Each six-membered ring is formed by six 1,3, 5-tri (4-aminophenyl) benzenes and six 2, 5-divinyl-1, 4-benzenedicarboxaldehydes arranged at intervals by covalent bonds. The theoretical mesopore size of the COF layer of the honeycomb structure is about 3.9 nm. Each 2, 5-divinyl-1, 4-benzenedicarboxaldehyde has two carbon-carbon double bonds as reaction sites for a thiol-ene click reaction.
From the above analysis, it is found that Fe provided by the present invention3O4The surface of the/COFs nanoparticle can modify more zwitter-ion GSH, so that the hydrophilic performance of the magnetic covalent organic framework material is improved to a great extent.
EXAMPLE 1 preparation of superparamagnetic Fe by hydrothermal method3O4Nano magnetic ball
Fe used in the following examples3O4The specific preparation process of the nano magnetic ball comprises the following steps: 2.43g of FeCl as a raw material3·6H2O, 3.6g NaAc (sodium acetate) and 0.4g Na3Adding CT (sodium citrate) into a reaction kettle containing 60mL of glycol, and stirring for 1 hour by magnetic force to uniformly mix the raw materials; then removing the stirrer, heating the reaction kettle to 200 ℃, and reacting for 12 hours; cooling the reaction kettle to room temperature, carrying out magnetic separation on the reaction liquid, and collecting the separated solid product; then washing the solid product with ethanol and deionized water in sequence (washing each washing solution for five times) to obtain Fe3O4And (4) nano magnetic balls.
Fe obtained by the method3O4The nano magnetic spheres can be uniformly dispersed in water to form stable superparamagnetic nanoparticle suspension. For the obtained Fe3O4DLS (Dynamic Light Scattering) analysis of the nano-magnetic spheres shows that the Fe is3O4The magnetic sphere has a particle size of about 220 nm.
The obtained Fe can be adjusted by adjusting the hydrothermal reaction time for 10-16 h3O4The particle diameter of the nano magnetic ball is 200-30Between 0 nm.
Example 2-example 8 preparation of Fe3O4/COFs nanoparticles (220 nm Fe is selected)3O4Nanometer magnetic ball)
The raw materials were weighed according to Table 1 and Fe was prepared according to the following procedure in combination with the process parameters given in Table 13O4PerFs nanoparticles:
mixing Fe3O4Adding two organic ligands of nano magnetic spheres, 1,3, 5-tri (4-aminophenyl) benzene (Tab) and 2, 5-divinyl-1, 4-benzene dicarbaldehyde (Dva) into a conical flask containing a dimethyl sulfoxide solvent, completely dissolving the two organic ligands under ultrasonic conditions, and allowing Fe to exist3O4And completely dispersing the nano magnetic spheres to obtain a uniformly dispersed mixed solution, then dropwise adding acetic acid into the obtained mixed solution under an ultrasonic condition, and standing and incubating the whole reaction system at room temperature after dropwise adding. Finally, carrying out magnetic separation on the obtained reaction liquid and collecting the separated solid product, and washing the obtained solid product with anhydrous tetrahydrofuran, anhydrous methanol, ethanol and deionized water in sequence (washing each washing solution for three times) to obtain Fe3O4/COFs nanoparticles. For the obtained Fe3O4DLS (Dynamic Light Scattering) analysis of the/COFs nanoparticles revealed that the resulting Fe3O4The particle size of the/COFs nanoparticles is about 250-670 nm, and is shown in Table 1.
TABLE 1 preparation of Fe3O4Raw materials of/COFs nano particles and proportion and process parameters thereof
As can be seen from Table 1, the encapsulation in Fe can be adjusted by adjusting the ratio of the two organic ligands, the ultrasound time and the incubation time3O4The thickness of the COF middle layer on the surface of the nano magnetic ball and the appearance of the nano composite material. The 270nm Fe prepared by the method described in example 2 was selected in consideration of the coating firmness, the size, the morphology of the nanocomposite, and the drug saving of the COF interlayer3O4the/COFs nanoparticles were used in the following examples Fe3O4Preparation of a/COF-GSH magnetic covalent organic framework material.
Example 9-example 16 preparation of Fe3O4The material is/COF-GSH (270 nm Fe is selected)3O4/COFs nanoparticles)
The following examples 9 to 16 employ the mercaptoene click reaction method using a two-neck flask reaction vessel heated by an oil bath and magnetically stirred as the reaction apparatus.
The raw materials were weighed according to Table 2 and Fe was prepared according to the following procedure in combination with the process parameters given in Table 23O4The material of/COF-GSH:
azodiisobutyronitrile, glutathione and Fe3O4the/COFs nano-particles are sequentially added into a composite solvent containing 20mL (the composite is obtained by uniformly mixing ethanol and deionized water according to the volume ratio of 1: 3), and the Fe is enabled to be processed by ultrasonic treatment3O4the/COFs nano particles are completely dispersed, and the azobisisobutyronitrile and the glutathione are completely dissolved. Then, deoxidizing the mixed solution for 1 hour by adopting a nitrogen purging mode; then, the branch-mouth bottle as the reactor was deoxygenated by a cyclic operation of vacuum-nitrogen introduction (cycle number 3 times). And then under the protection of nitrogen, adding the deoxygenated mixed solution into a branched bottle from which aerobic gas is removed in advance, then obtaining the reaction temperature shown in the table 2 through oil bath, and completing the mercaptoalkene click reaction according to the reaction temperature and the reaction time shown in the table 2 under the magnetic stirring. Finally, carrying out magnetic separation on the obtained reaction liquid and collecting the separated solid product, and washing the obtained solid product with ethanol and deionized water in sequence (washing each washing solution for three times) to obtain Fe3O4a/COF-GSH material. For the obtained Fe3O4The DLS (dynamic light Scattering) analysis of the/COF-GSH material is used for investigating the dispersion performance in water: the more successful the GSH modification is, Fe3O4The better the dispersion performance of the/COF-GSH material in water, the smaller the PDI value of the DLS test.
TABLE 2 preparation of Fe3O4Raw materials of/COF-GSH material and proportion and process parameters thereof
As can be seen from Table 2, the optimum GSH modification was achieved by controlling the amount of zwitterionic GSH and AIBN added, the reaction temperature and the reaction time. Now, the scheme of example 12 was selected to prepare Fe in consideration of modification effect, reaction time saving, and raw material saving3O4The structure characteristics, the performance, the glycopeptide enrichment and the like of the/COF-GSH material are researched.
Structural characterization
To investigate whether COFs are successfully complexed to Fe3O4On a nanomagnetic sphere, for Fe prepared in example 13O4Nano-magnetic spheres, Fe prepared in example 23O4CoFs nanoparticles, Fe prepared in example 123O4The morphological dimensions and microstructure of the/COF-GSH magnetic covalent organic framework material are characterized as shown in FIGS. 2 to 5.
For Fe prepared in example 23O4CoFs nanoparticles and Fe prepared in example 123O4the/COF-GSH magnetic covalent organic framework material is subjected to morphology analysis by adopting a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM), and the result is shown in figure 2. As can be seen from FIG. 2, Fe was produced3O4CoFs nanoparticles and Fe3O4the/COF-GSH materials are all spherical with uniform size and regular appearance. In addition, as seen from fig. 2E, the surface of the nanoparticle presents a rough network structure, indicating that the COFs layer has been successfully wrapped in Fe3O4The surface of the nano magnetic ball; as seen in FIG. 2F, Fe3O4/COF-GSH material and Fe3O4the/COFs nano-particles have almost the same appearance, and are subjected to surface modification of zwitter ion GSH and then are used for Fe3O4The morphology of the/COFs nanoparticles has no obvious influence.
For Fe prepared in example 13O4Nano-magnetic spheres, Fe prepared in example 23O4CoFs nanoparticles and Fe prepared in example 123O4The analysis result of the/COF-GSH material by X-ray diffraction is shown in figure 3. As can be seen from FIG. 3, all three materials have Fe equivalent to the standard3O4The characteristic peak of the diffraction peak is consistent, which indicates that Fe3O4the/COF-GSH material retains Fe3O4Crystal structure of nano magnetic ball. While Fe3O4the/COF-GSH material has a new diffraction peak at 2.7 degrees, and the diffraction peak is a characteristic diffraction peak of the covalent organic framework, which indicates that the covalent organic framework is successfully synthesized on the surface of the magnetic nanoparticle.
For Fe prepared in example 123O4The results of X-ray energy spectrum analysis (EDX) of the/COF-GSH magnetic covalent organic framework material are shown in FIG. 4 and Table 3. From FIG. 4 and Table 3, Fe3O4In the/COF-GSH magnetic covalent organic framework material, the atomic percent of C element is 55.97%, the atomic percent of N element is 15.52%, the atomic percent of O element is 23.28%, the atomic percent of S element is 2.01%, and the atomic percent of Fe is 3.22%. As can be seen from the above results, Fe3O4The existence of S element and high content of N element in the/COF-GSH material further proves that the COFs layer and the zwitter ion GSH have been successfully applied to Fe3O4The surface of the nano magnetic sphere is formed and modified, and the high-content GSH is beneficial to enrichment application of glycopeptide.
TABLE 3 atomic percentages of the elements obtained in the EDX analysis
| Element | Weight％ | Atomic％ |
| C | 44.63 | 55.97 |
| N | 14.43 | 15.52 |
| o | 24.72 | 23.28 |
| S | 4.29 | 2.01 |
| Fe | 11.92 | 3.22 |
| Total | 100 | 100 |
Fe prepared in example 2 and example 123O4CoFs nanoparticles and Fe3O4N on/COF-GSH materials2Adsorption/desorption test, the test results are shown in FIG. 5, and the obtained N2The adsorption/desorption isothermal curve shows that the magnetic covalent organic framework nano composite material has larger specific surface area and porosity, and the pore size distribution is narrower and is about 3.6nm (similar to a theoretical analysis result), which indicates that the covalent organic framework of the mesoporous material with high order is successfully synthesized on the surface of the magnetic sphere, and the enrichment application of the peptide fragment is facilitated.
In conclusion, Fe3O4The shell layer in the/COF-GSH material has a covalent organic framework structure, and the structure does not obviously influence the magnetic crystal structure of the composite nano material, so that the unique COF shell layer is beneficial to the application in glycopeptide capture and separation.
Magnetic property test
For Fe prepared in example 13O4Nano-magnetic spheres, Fe prepared in example 23O4CoFs nanoparticles, Fe prepared in example 123O4The magnetic property of the/COF-GSH material is tested in a range from-18000 Oe to 18000Oe by adopting a Model BHV-525 Vibration Sample Magnetometer (VSM), and a hysteresis loop obtained by the test is shown in figure 6. As can be seen from FIG. 6, the hysteresis loops of all samples pass through the origin, there is no remanence and coercivity, and the description shows Fe3O4Nano magnetic ball, Fe3O4/COFs nanoparticles, Fe3O4the/COF-GSH materials all have superparamagnetism, wherein Fe3O4The saturation magnetization of the/COF-GSH material reaches 45emug-1Left and right.
Application example
The present invention further provides the above Fe3O4Application of/COF-GSH material in glycopeptide enrichment, Fe3O4The capture and separation process of the/COF-GSH material for glycopeptide is shown in FIG. 1B, and Fe is firstly carried out3O4Adding the/COF-GSH material into a sample to be processed, then capturing in a shaking table, wherein the time can be adjusted according to the amount of the sample, separating a solid product by a magnetic separation mode after the capturing process is finished, and then desorbing the magnetic ball with the glycopeptide adsorbed on the surface by using a desorbed buffer solution, thereby obtaining the glycopeptide-containing buffer solution. The resulting glycopeptide-containing buffer can be subjected to MS (Mass Spectrometry) analysis to further determine Fe3O4The enrichment effect of the/COF-GSH material on glycopeptide.
Application example 1 enrichment of glycopeptides in a glycosylated protein immunoglobulin G digest
2mg of immunoglobulin G was dissolved in 1ml of NH 50mM pH 8.24HCO3Adding 50 μ g of trypsin into the buffer solution, and digesting for 16h at 37 ℃; then using a first buffer (90% acetonitrile aqueous solution containing 1% trifluoroacetic acid by volume, i.e. 90% ACN-H)2O contains 1% TFA) to 10-7Concentration of MObtaining the immunoglobulin G digestive juice. 1mg of Fe prepared in example 12 was taken3O4Adding the/COF-GSH material into 200 μ l of immunoglobulin G digestive juice sample, and then incubating for 45min at room temperature under the conditions of a shaking table 150 and 200 rpm; then washing 3 times (200. mu.l each) with a first buffer solution to remove the non-specifically adsorbed polypeptides from the surface of the magnetic covalent organic framework material; finally, the magnetic covalent organic framework material adsorbed with glycopeptide is added into 10. mu.l of a second buffer (30% by volume acetonitrile aqueous solution containing 0.1% by volume trifluoroacetic acid, i.e., 30% ACN-H)2O comprises 0.1% TFA), desorbing for 30min under shaking at shaking table 800-1200rpm, and separating out magnetic covalent organic framework material by magnetic separation to obtain desorbed solution. Then, 1. mu.l of the desorption solution and 1. mu.l of the digestion solution of immunoglobulin G which has not been subjected to enrichment treatment were subjected to mass spectrometry, and the results of the analysis are shown in FIG. 7.
As can be seen from FIG. 7, the immunoglobulin G digestion solution without enrichment treatment only obtained 5 glycopeptide signals by mass spectrometry, and the signals were very low, and the whole MS graph was basically the signal of the hetero peptide fragment (see FIG. 7); with Fe3O4After enrichment of the/COF-GSH material, signals of 35 glycopeptides can be detected on the whole mass spectrogram, and the whole MS diagram is a signal peak of the glycopeptides (see figure 7, and the round dots are all characteristic peaks of the glycopeptides). The above analysis results show that Fe according to the present invention3O4the/COF-GSH material can be used for enriching glycopeptides well, and has good selectivity and high efficiency.
Application example 2 enrichment of endogenous glycopeptides in human saliva
10 microliters of healthy human saliva was added to 200. mu.l of a first buffer (90% by volume acetonitrile in water containing 1% by volume trifluoroacetic acid, i.e., 90% ACN-H)2O contained 1% TFA). 1mg of Fe prepared in example 12 was taken3O4Adding the/COF-GSH material into the sample, and then incubating for 45min at room temperature under the conditions of a shaking table 150 and 200 rpm; then washing 3 times (200. mu.l each) with a first buffer solution to remove the non-specifically adsorbed polypeptides from the surface of the magnetic covalent organic framework material; finally, the magnetism adsorbed with glycopeptide is combinedThe organic framework material was added to 60. mu.l of a second buffer (30% by volume aqueous acetonitrile containing 0.1% by volume trifluoroacetic acid, i.e., 30% ACN-H)2O comprises 0.1% TFA), desorbing for 30min under shaking at shaking table 800-1200rpm, and separating out magnetic covalent organic framework material by magnetic separation to obtain desorbed solution. The adsorption liquid was then used for LC-MS analysis, and the results are shown in table 4, where a total of 322 endogenous glycopeptides could be detected.
Table 5 shows Fe provided by the present invention3O4the/COF-GSH material is compared with the reported material on the enrichment performance of endogenous glycopeptides in human saliva. As seen from Table 5, the present invention provides Fe in comparison with the reported materials3O4the/COF-GSH material can be enriched in the most endogenous glycopeptides in human saliva.
In summary, the Fe of the present invention3O4the/COF-GSH magnetic covalent organic framework material can realize excellent enrichment performance on glycopeptides and endogenous glycopeptides in human saliva.
TABLE 4 Fe3O4@ COF-GSH magnetic covalent organic framework material
Detailed information of endogenous glycopeptides enriched from human saliva
TABLE 5 Fe3O4Comparison of performance of/COF-GSH magnetic covalent organic framework material and reported material in enriching endogenous glycopeptide in human saliva
Claims (10)
1. A magnetic covalent organic frame material for enriching glycopeptide is prepared from Fe3O4Nano magnetic ball coated on Fe3O4The covalent organic framework layer on the surface of the nano magnetic ball and the glutathione grafted on the covalent organic framework layer; the covalent organic framework layer is prepared by mixing 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde according to the mass ratio of 1:1.5 a cellular structure obtained by an addition reaction, the glutathione being grafted on the vinyl groups of the covalent organic framework layer.
2. The magnetic covalent organic framework material of claim 1, wherein the magnetic covalent organic framework material is in the form of spherical particles with an average particle size of 240-300 nm.
3. The method for preparing a magnetic covalent organic framework material for glycopeptide enrichment according to claim 1 or 2, characterized by the following steps:
(1) preparation of Fe3O4/COFs nanoparticles
Under the ultrasonic condition, Fe3O4Mixing and dispersing nanometer magnetic spheres, 1,3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-benzene dicarbaldehyde in dimethyl sulfoxide uniformly to form a mixed solution; then, under the ultrasonic condition, dropwise adding acetic acid into the mixed solution to form a reaction system; then, standing and incubating the obtained reaction system at room temperature for 10-30 minutes, carrying out magnetic separation on the obtained reaction liquid after incubation is finished, collecting separated solid products, washing the obtained solid products to remove unreacted materials adsorbed on the surfaces of the solid products, and obtaining covalent organic framework layer coated Fe3O4Nanoparticles of nano-magnetic spheres, abbreviated as Fe3O4/COFs nanoparticles; said Fe3O4The mass ratio of the nano magnetic ball to the 1,3, 5-tri (4-aminophenyl) benzene is 1: (0.2-1), the mass ratio of 1,3, 5-tri (4-aminophenyl) benzene to 2, 5-divinyl-1, 4-benzene dicarbaldehyde is 1:1.5, and the volume ratio of the mass sum of the 1,3, 5-tri (4-aminophenyl) benzene and the 2, 5-divinyl-1, 4-benzene dicarbaldehyde to acetic acid is (30-210): 1, the unit of mass is mg, and the unit of volume is mL;
(2) preparation of Fe3O4/COF-GSH material
Azodiisobutyronitrile, glutathione and Fe3O4the/COFs nano particles are sequentially added into a composite solvent to form a mixed solution, and then under the protection of nitrogen, the obtained mixed solution is subjected to mercaptoene click reaction at the temperature of 60-80 ℃ until Fe is obtained3O4The surface of the COFs nanoparticle is yellow; after finishing mercaptoene click reaction, carrying out magnetic separation on the obtained reaction liquid, collecting the separated solid product, washing the obtained solid product to remove unreacted materials adsorbed on the surface of the solid product, and drying to obtain the magnetic covalent organic framework material for glycopeptide enrichment, Fe for short3O4a/COF-GSH material; the composite solvent is obtained by uniformly mixing ethanol and deionized water according to the volume ratio of 1:3, and Fe is contained in the mixed solution3O4The mass ratio of the/COFs nano particles to the glutathione is 1 (0.3-1); the mass ratio of the glutathione to the azodiisobutyronitrile is 1 (0.05-0.25).
4. The method for preparing a magnetic covalent organic framework material for glycopeptide enrichment according to claim 3, wherein in the step (1), the acetic acid is added dropwise for 5-15 min.
5. The method according to claim 3 or 4, wherein in the step (2), the mixture solution is subjected to an oxygen removal treatment to remove oxygen from the mixture solution and above the mixture solution in the reactor before the sulfydryl click reaction under the protection of nitrogen.
6. The method of claim 5, wherein the oxygen scavenging treatment is performed by: firstly, deoxidizing the mixed solution by adopting a nitrogen purging mode; and then removing oxygen above the mixed liquid in the reactor by adopting a circulation mode of vacuumizing and introducing nitrogen.
7. The method for preparing magnetic covalent organic framework material for glycopeptide enrichment according to claim 3 or 4, wherein the solid product in step (1) is sequentially washed with anhydrous tetrahydrofuran, anhydrous methanol, ethanol and deionized water, and the solid product in step (2) is sequentially washed with ethanol and deionized water.
8. The method according to claim 5, wherein the solid product of step (1) is sequentially washed with anhydrous tetrahydrofuran, anhydrous methanol, ethanol, and deionized water, and the solid product of step (2) is sequentially washed with ethanol and deionized water.
9. The method according to claim 6, wherein the solid product of step (1) is sequentially washed with anhydrous tetrahydrofuran, anhydrous methanol, ethanol, and deionized water, and the solid product of step (2) is sequentially washed with ethanol and deionized water.
10. Use of the magnetic covalent organic framework material of claim 1 or 2 for enrichment of endogenous glycopeptides.
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