CN109721759B - Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment - Google Patents
Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment Download PDFInfo
- Publication number
- CN109721759B CN109721759B CN201711035056.0A CN201711035056A CN109721759B CN 109721759 B CN109721759 B CN 109721759B CN 201711035056 A CN201711035056 A CN 201711035056A CN 109721759 B CN109721759 B CN 109721759B
- Authority
- CN
- China
- Prior art keywords
- acetylglucosamine
- acid
- functional monomer
- initiator
- acetyl glucosamine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention relates to an acetylglucosamine (N-GlcNAc) imprinted material and application thereof in recognition of acetylglucosamine and an acetylglucosamine modified peptide fragment. The imprinting material is prepared by using acetylglucosamine as a template molecule and polymerizing a functional monomer and a cross-linking agent, and is used for recognizing acetylglucosamine and a modified peptide thereof.
Description
Technical Field
The invention belongs to a preparation technology of a high polymer material and application thereof in biomolecule recognition, and particularly relates to a novel acetylglucosamine imprinted material, and preparation and application thereof.
Background
Acetylglucosamine modification is a post-translational modification of proteins that is ubiquitous in mammals. Numerous studies have demonstrated that acetylglucosamine modifications play an important role in cell structure, cell cycle, regulation of cellular metabolism and transcription, and nuclear substance transport. In addition, acetylglucosamine modification and oxyphosphorylation modification can competitively occur at the same serine or threonine site, and the mutual site occupation condition can cause different activity or stability changes of the protein, thereby realizing the regulation and control of cell signaling pathways and biological processes thereof by utilizing the form of interactive dialogue. Current studies have demonstrated that modification of acetylglucosamine modification is associated with a variety of diseases, such as cancer and diabetes. Therefore, the research of the posttranslational modification of the acetylglucosamine has important significance.
However, the characteristics of low abundance, dynamic modification and the like of the acetylglucosamine modified protein bring great challenges to the research thereof. In order to overcome the challenge of low abundance, the acetylglucosamine modified peptide fragment needs to be enriched at the stage of sample pretreatment, and the currently reported methods comprise a lectin affinity enrichment method, an antibody enrichment method, a chemical labeling method, an enzyme-catalyzed labeling method, a boron affinity enrichment method and the like. However, the above methods have the disadvantages of low affinity, low specificity, and easy generation of false positive.
The molecular imprinting technology is a preparation technology for obtaining a high molecular polymer with a spatial structure, a size and a binding site which are completely matched with template molecules by polymerizing a functional monomer and a cross-linking agent in the presence of the template molecules. Because the molecular imprinting sites formed in the imprinting process generally have affinity and selectivity similar to those of an antigen-antibody system, the molecular imprinting sites have high affinity and selectivity for target molecules. Meanwhile, the MIP has the predetermination, and the corresponding MIP is easily synthesized according to the structural design of different target molecules. Finally, MIP as a high molecular polymer has higher stability than biomolecules such as antibodies, and can be used in a wide range of chemical and physical environments.
However, as a biomacromolecule, proteins are large in molecular weight, bulky, and sensitive to environmental changes in their conformation; meanwhile, for many proteins with important biological significance, it is difficult to obtain enough pure protein as a template to prepare the blotting material. An epitope is a region of an antigen surface that determines its immunogenicity, and capture of an antigen by an antibody is through recognition of its epitope by the antibody. Based on this, researchers prepared blots with the terminal peptide of the target protein as the epitope and the epitope as the template. This recognition pattern is similar to the recognition of an antigen by an antibody through an epitope and is therefore referred to as an epitope blot. The epitope imprinting material has the advantage of common recognition of target proteins and peptide fragments with the same epitope, and is primarily applied to enrichment of phosphorylated peptide fragments and acetylated peptide fragments.
In order to overcome the problems of low enrichment affinity and insufficient specificity of the acetylglucosamine modified peptide fragment, an epitope imprinting technology is adopted, a characteristic fragment of the acetylglucosamine modified peptide fragment is selected as a template, and a molecular imprinting method is adopted to prepare an acetylglucosamine imprinting material which is used for recognizing the acetylglucosamine and the acetylglucosamine modified peptide fragment.
Disclosure of Invention
Selecting acetylglucosamine as a template molecule by utilizing an epitope imprinting technology, preparing an acetylglucosamine imprinting material by adopting a body imprinting and Pickering emulsion imprinting technology, and removing the template to form the acetylglucosamine imprinting material; and the imprinted material is used for identifying the acetylglucosamine and the acetylglucosamine modified peptide fragment. In order to achieve the purpose, the invention adopts the technical scheme that:
1. mixing the acetylglucosamine, the functional monomer, the cross-linking agent, the initiator and the solvent, polymerizing, and removing the template molecules to obtain the acetylglucosamine bulk imprinting material. The method comprises the following specific steps:
(1) dissolving acetyl glucose, adding a functional monomer, a cross-linking agent and an initiator, and dissolving and uniformly mixing all the components by ultrasonic or oscillation to form a prepolymerization solution.
(2) The polymerization is initiated at high temperature (above 50 deg.C), UV, or redox conditions to promote polymerization of the pre-polymerized solution.
(3) And (3) eluting the obtained polymer by using a template removal solvent, and removing template molecules to form the acetylglucosamine bulk imprinting material.
(4) The non-imprinted polymer was prepared by the same procedure as above without adding acetylglucosamine to the prepolymerization solution.
(5) The bulk imprinted polymer is used for recognition of acetylglucosamine and modified peptide fragments thereof.
The acetylglucosamine dissolving solvent is characterized in that: one or more of water, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, chloroform, tetrahydrofuran, dimethyl sulfoxide, dimethylacetamide, and dimethylformamide.
In the bulk imprinting system, the mass fraction of the acetylglucosamine in the total system is 0.1-40%; the mass fraction of the functional monomer in the total system is 5-90%; the mass fraction of the cross-linking agent in the total system is 5-45%; the mass fraction of the solvent in the total system is 0.01-10%.
The functional monomer is as follows: monomers capable of polymerizing to form a high molecular polymer include one or more of acrylamide, methacrylamide, isopropylacrylamide, hydroxyethylacrylamide, phenylpropenamide, acrylic acid, methyl acrylate, butyl acrylate, methacrylic acid, methyl methacrylate, ethyl methacrylate, butyl methacrylate, tetramethoxysilane, aminotrimethoxysilane, vinyltrimethoxysilane, butyltrimethoxysilane, trimethoxysilane phenylboronic acid, tetraethoxysilane, aminotriethoxysilane, vinyltriethoxysilane, butyltriethoxysilane, triethoxysilane phenylboronic acid, dopamine, vinylpyrrolidone, vinylimidazole, vinyloxirane, styrene, vinylphenylboronic acid, vinylbenzoic acid, methoxypropene, and allyl methyl ether.
The cross-linking agent is as follows: one or more of methylene bisacrylamide and ethylene glycol dimethacrylate.
The initiator refers to: persulfate initiator, azobisisobutyronitrile, azobisisoheptonitrile, redox initiator such as ammonium persulfate/sodium bisulfite, potassium persulfate/sodium bisulfite, etc.
The solvent for removing the template is as follows: alcohol solvent or alcohol acetic acid solution, wherein the volume concentration of acetic acid in the alcohol acetic acid solution is 0-50%; the template removing method is that the imprinting materials are sequentially eluted by using a template removing solvent.
2. Solid particles bonded with acetylglucosamine groups are used as a stabilizer, mixed liquid of a water phase and an oil phase is added to form stable Pickering emulsion, and the stabilizer and the template are removed after polymerization is initiated to form the acetylglucosamine imprinted microspheres. The method comprises the following specific steps:
(1) acetyl glucosamine is bonded on the surface of solid particles such as silicon matrix, metal oxide matrix and the like by utilizing covalent bonds and is used as a stabilizer of the pickering emulsion.
(2) Mixing a water phase containing a water-soluble functional monomer and a water solvent, an oil phase containing an oil-soluble functional monomer, a cross-linking agent, an initiator and an oil solvent, and a stabilizer, and preparing the stable pickering emulsion by using an ultrasonic or shaking mode.
(3) Polymerization is initiated under high temperature (higher than 50 ℃), ultraviolet or oxidation-reduction conditions to promote the polymerization reaction of the Pickering emulsion oil phase.
(4) Dissolving the stabilizer matrix material by using strong acid or strong base to remove the stabilizer; and then removing the solvent eluting material by using the template, and removing the template molecules to form the acetylglucosamine surface imprinted microspheres.
(5) The non-imprinted microspheres are prepared by the same procedure as above using solid particles to which acetylglucosamine is not bonded as a stabilizer.
(6) The surface imprinted microspheres are used for recognition of acetylglucosamine and modified peptide fragments thereof.
The acetyl glucosamine linkage refers to: forming covalent bond between hydroxyl on the acetyl glucosamine and the matrix material; or other reactive groups (including alkynyl, azide, alkenyl, sulfhydryl, amino, carboxyl, epoxy, etc.) can be introduced on the acetylglucosamine to bond the acetylglucosamine to the matrix material. The solid particles refer to: silicon matrix or metal oxide matrix particles between 1 nanometer and 1 micrometer.
The aqueous phase functional monomer refers to: acrylamide and acrylic acid functional monomers, including acrylamide, methacrylamide, isopropyl acrylamide, hydroxyethyl acrylamide, phenylpropenamide, acrylic acid, methyl acrylate, butyl acrylate, methacrylic acid, methyl methacrylate, ethyl methacrylate and butyl methacrylate, with the mass concentration of 1-20%; the mass concentration of the stabilizer is 1-15%. The oil phase refers to: wherein the oil-soluble functional monomer comprises vinyl pyrrolidone, vinyl imidazole, vinyl oxirane, styrene, vinyl phenylboronic acid, vinyl benzoic acid, methoxy propylene and allyl methyl ether, the cross-linking agent comprises methylene bisacrylamide, ethylene glycol dimethacrylate and the like, the initiator is azo initiator, such as azobisisobutyronitrile, azobisisoheptonitrile, azobisisobutylamidine hydrochloride and the like, and the solvent comprises toluene, chloroform, acetone, tetrahydrofuran and the like. The mass fraction of the functional monomer in the total system is 0-30%; the mass fraction of the cross-linking agent in the total system is 10-99%; the mass fraction of the initiator in the total system is 0.01-10%; the mass fraction of the solvent in the total system is 0-50%.
The stabilizer is removed by strong acid or strong alkali, and the method comprises the following steps: the stabilizer is removed by dissolving the matrix material with hydrofluoric acid, ammonium hydrofluoride, hydrochloric acid, sulfuric acid, etc. at a concentration of 500mM-5M, or sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, etc. at a concentration of 500 mM-5M.
The solvent for removing the template is as follows: alcohol solvent or alcohol acetic acid solution, wherein the volume concentration of acetic acid in the alcohol acetic acid solution is 0-50%; the template removing method is that the imprinting materials are sequentially eluted by using a template removing solvent.
The invention has the following advantages:
(1) the invention utilizes the acetylglucosamine as a template molecule to prepare the imprinting material, the acetylglucosamine has low price and is easy to purchase, and the problem that the template is difficult to obtain in the biomolecular imprinting is solved.
(2) The acetylglucosamine is a common characteristic fragment of all the acetylglucosamine modified peptide fragments, so that the imprinted material prepared by taking the acetylglucosamine as a template molecule can realize the recognition of all the acetylglucosamine modified peptide fragments, and the recognition process has universality.
(3) The molecularly imprinted microsphere prepared by the Pickering emulsion imprinting method by using the solid particles bonded with the acetylglucosamine groups as the stabilizer has the imprinting sites on the surface of the microsphere, and has the advantages of easy template elution, small mass transfer resistance and rapid target molecule adsorption.
(4) The method utilizes a Pickering emulsion method to prepare the imprinted microspheres, and can effectively regulate and control the size of the microspheres through the addition of the stabilizer, thereby meeting the requirements of different application requirements on the particle size.
(5) The invention can adopt a plurality of different functional monomers to polymerize to form the imprinting sites. The utilization of various functional monomers can provide various abundant interactions to a great extent, and ensure the specific recognition of the formed imprinting sites to the acetylglucosamine and the modified peptide fragments thereof.
(6) The imprinting material can specifically identify the acetylglucosamine and the modified peptide fragment thereof, and has important application value in the separation and purification of the acetylglucosamine and the enrichment identification of the acetylglucosamine modified peptide fragment.
Description of the drawings:
FIG. 1 is a graph of adsorption capacity of acetylglucosamine bulk imprinted material (MIP1) for acetylglucosamine.
FIG. 2 is a morphology chart of an acetyl glucosamine surface imprinted material (MIP2) prepared by Pickering emulsion imprinting. (A) The scale of the graph is 10 microns, and the scale of the graph (B) is 1 micron.
FIG. 3 is a chromatographic separation diagram of a surface imprinted material (MIP2) and a non-imprinted material (NIP2) of the acetylglucosamine prepared by Pickering emulsion imprinting method on the acetylglucosamine.
FIG. 4 shows the effect of MIP2 on the enrichment of acetylglucosamine modified peptide fragments in the enzymatic hydrolysate of rat brain tissue extract.
FIG. 5 is a topographical representation of a boron affinity assisted acetylglucosamine surface imprinted material (MIP 3). (A) The scale of the graph is 10 microns, and the scale of the graph (B) is 1 micron.
FIG. 6 is a graph of the adsorption capacity of boron affinity-assisted acetylglucosamine surface imprinted material (MIP3) and non-imprinted material (NIP3) to acetylglucosamine.
Fig. 7 is a graph showing adsorption capacities of MIP3 for acetylglucosamine, glycine (Gly), and serine (Ser).
FIG. 8 shows the effect of MIP3 on the enrichment of acetylglucosamine modified peptide fragments in the enzymatic hydrolysate of rat brain tissue extract.
Detailed Description
Example 1
Preparation of acetylglucosamine bulk imprinted material (MIP1)
35mg of N-acetyl-D-glucosamine was dissolved in 0.5mL of dimethyl sulfoxide, 300. mu.L of ethylene glycol dimethacrylate, 50mg of acrylamide and 3.5mg of azobisisobutyronitrile were added, and after all the components were completely dissolved, nitrogen was introduced into the reaction mixture for 10 min. The mixture was allowed to stand at 4 ℃ for 1 hour and then reacted at 65 ℃ for 12 hours. The bulk material obtained by the reaction was removed, and after grinding with a mortar, the template was eluted 10 times for 5 hours each time by adding 4mL of a template-removing solution methanol: acetic acid (9: 1). Finally, the obtained material is sieved by a 300-mesh screen, particles smaller than 300 meshes are reserved, and the acetyl glucosamine bulk imprinted material (MIP1) is obtained after drying.
In the formula, the mixing mass ratio of the acetyl glucosamine, the functional monomer, the cross-linking agent, the initiator and the solvent is 4:5.6:33.8:0.4: 56.2.
Non-imprinted microspheres (NIP) were prepared in the same way, but without the addition of acetylglucosamine.
20mg of MIP1 or NIP1 were added to 400. mu.L of 0.1mg/mL glucosamine dissolved in acetonitrile at different concentrations and incubated for 24h at 25 ℃. Centrifuging at 2500rpm for 5min, and removing supernatant; and the material was washed 2 times with 200 μ L of 80% acetonitrile; finally, the adsorbed acetylglucosamine was eluted by the material 2 times for 12 h/time with 80. mu.L of methanol: acetic acid (9: 1); the eluate was collected and purified by high performance liquid chromatography (Hilic high purity chromatography, HILIC Click Xlon column 4.6X 250mm,5 μm,
) And detecting the concentration of the acetylglucosamine in the eluent by a peak area quantitative method, and further calculating to obtain the adsorption capacity.
As shown in fig. 1, the adsorption capacity of MIP1 for acetylglucosamine was higher than that of NIP1 at different acetonitrile concentrations, indicating that MIP1 had higher affinity for acetylglucosamine than NIP1 due to the presence of the imprinted site. The blotting factor was highest at 85% acetonitrile as incubation solvent, and reached 1.7, at which time the adsorption capacity of MIP1 for acetylglucosamine was 0.05 mg/g.
Example 2
Pickering emulsion blotting method for preparing acetyl glucosamine surface blotting material (MIP2)
The boric acid modified silicon nano-particles are used as a matrix material, and the particle size is 30 nm. 100mg of acetylglucosamine is weighed and dissolved in 1mL of dimethyl sulfoxide, 100mg of boric acid modified silicon nano particles are added, and the mixture is subjected to ultrasonic treatment for 10min and then subjected to oscillation reaction at 60 ℃ for 3 h. Centrifuging at 17000rpm for 20min, collecting reaction product, washing with pure water, preparing silicon nanoparticles bonded with acetylglucosamine, freeze drying for 50min, and storing for use as stabilizer of Pickering emulsion.
50mg of silicon nano particles bonded with acetylglucosamine are ultrasonically dispersed in 2.5mL of water, 68mg of functional monomer acrylamide is added, and after the functional monomer acrylamide is completely dissolved, the mixture is kept stand for 50min at 4 ℃ to be used as the water phase of the Pickering emulsion. Adding 15mg of azodiisobutyronitrile into 0.342mL of ethylene glycol dimethacrylate, and adding the solution into a 10mL glass bottle after ultrasonic dissolution; then adding the water phase after standing into a glass bottle, and shaking vigorously for 30s by hand; standing on ice for 2min, and shaking vigorously for 1 min; standing again on ice for 2min, and shaking vigorously for 1 min; standing at 4 deg.C for 50min, and reacting at 70 deg.C for 12 h.
Transferring the material obtained by polymerization into a centrifugal tube, centrifuging at 1250rpm for 5min, collecting the bottom material of the tube, cleaning the material with ultrapure water for 3 times, and cleaning with methanol for 1 time. Then 3mL of methanol/hydrofluoric acid (v/v, 2:1) was added, the material was etched at room temperature for 2h, collected, and etched again with 3mL of the etching solution for 2h, so that the silicon substrate was completely dissolved and removed. And finally, sequentially washing the substrate with methanol-acetic acid (9:1, V: V) for 5 times, washing the substrate with ultrapure water for 2 times, washing the substrate with methanol for 2 times, and drying the substrate in vacuum to obtain the acetylglucosamine surface imprinted material (MIP 2).
The preparation process of the non-imprinted material (NIP2) is similar to that of MIP2, but the stabilizer is not added with acetyl glucosamine during the template fixing process.
As shown in fig. 2A, the acetylglucosamine surface imprinted material is standard spherical particles, and the surface is smooth; on a partial magnification (fig. 2B), a large number of recessed sites on the surface of MIP2 can be seen, as the surface stabilizer is dissolved away, indicating that hydrofluoric acid can remove the surface stabilizer and form imprinted sites.
Example 3
MIP2 recognition of acetylglucosamine and acetylglucosamine modified peptide fragments
700mg of MIP2/NIP2 was added to 40mL of homogenate of 50% acetonitrile and sonicated for 5min to form a uniform suspension. The column was packed under a pressure of 30MPa, using column tubes of 2.1X 150mm in size. After completion of the column packing, the column was washed with 50% acetonitrile at a flow rate of 0.1mL/min for 2h, followed by washing with 0.2mL/min 80% acetonitrile for 2h and stored until use.
By using Hitachi high-tech Chromaster chromatographic system, acetonitrile with different proportions is used as a mobile phase, the flow rate is 0.2mL/min, 5 mu L of 0.1mg/mL of acetylglucosamine is injected, the detection wavelength is 200nm, and the retention time (t) of the acetylglucosamine on MIP2 and NIP2 columns is measured (t)R). 5 μ L of 20% acetone was injected and the peak was observedAs the system dead time (t)0). By the formula k ═ tR-t0)/t0And calculating retention factor k of the acetylglucosamine on MIP2 and NIP2 columns, and using k as an evaluation index to explain the strength of the MIP2 and NIP2 on the acting force of the acetylglucosamine.
FIG. 3 shows the chromatographic separation of acetylglucosamine on MIP2 column and NIP2 column. It can be seen that at 80% acetonitrile as the mobile phase (fig. 3A), the retention of acetylglucosamine is very weak, with slightly stronger retention on the MIP2 column than on the NIP2 column, indicating that MIP2 has a stronger affinity for acetylglucosamine than NIP 2. When the acetonitrile concentration in the mobile phase was increased to 95%, the retention of acetylglucosamine was enhanced, and it was seen that the retention of acetylglucosamine on the MIP2 column was significantly stronger than that of NIP2, when the IF was 1.50 as calculated by the retention factor.
To further evaluate the enrichment performance of MIP2, it was used for enrichment of acetylglucosamine modified peptide fragments in the enzymatic hydrolysate of murine brain tissue. As shown in FIG. 4, a total of 12 acetylglucosamine modified proteins were identified from the original rat brain tissue hydrolysate, corresponding to 18 acetylglucosamine modified peptide fragments and 50 PSMs. After MIP3 enrichment, the number of the identified acetylglucosamine modified proteins is increased to 20, the number of the acetylglucosamine modified peptide fragments is increased to 50, and the number of corresponding PSMs is increased to 222, which indicates that MIP2 has better enrichment capacity on the acetylglucosamine modified peptide fragments.
Example 4
Preparation of boron affinity assisted acetyl glucosamine surface imprinted material (MIP3)
Boron affinity assisted acetyl glucosamine surface imprinted material (MIP3) and non-imprinted material (NIP3) were prepared similarly to example 2, except that different stabilizers were used and the way of preparing Pickering emulsion by ultrasound.
The preparation method of the stabilizer comprises the following steps: the mercapto-modified silicon nanoparticles are used as a matrix material of a stabilizer, and the particle size is 300 nm. 1g of mercapto-modified silicon nanoparticles and 500mg of 1-allyl-modified acetylglucosamine were added to 50mL of a methanol-water (1:1) solution; after 30min of ultrasonic treatment, 50mg of azobisisobutylamidine hydrochloride is added, nitrogen is introduced for 20min, and then the mixture is magnetically stirred and reacted for 22h at 65 ℃. Centrifuging at 17000rpm for 10min, collecting the product, and alternately washing with water and methanol for 2 times to obtain silicon nanoparticles bonded with acetylglucosamine.
200mg of silicon nanoparticles bonded with acetylglucosamine and 100mg of acrylamidophenylboronic acid were added to 1.5mL of dimethyl sulfoxide, and the mixture was subjected to ultrasonic treatment for 10min to completely disperse the silicon nanoparticles, followed by shaking reaction at 65 ℃ for 10 hours. Subsequently, centrifuging at 20000rpm for 30min, and collecting the centrifugation product; washing with 1mL of methanol for 2 times, and vacuum drying at 60 deg.C to obtain silicon nanoparticles bonded with acetylglucosamine and acetylphenylboronic acid, which are used as stabilizer for Pickering emulsion, drying, and storing.
The method for preparing the pickering emulsion by ultrasonic comprises the following steps: adding the water phase after standing into a glass bottle, and shaking vigorously for 30s by hand; standing on ice for 2min, and performing ultrasonic treatment for 1 min; standing on ice for 2min again, and performing ultrasonic treatment under 50% of energy; the template was pre-assembled with EGDMA by non-covalent interaction at 4 ℃ for 50 min.
As shown in figure 5, no demulsification phenomenon occurs in the Pickering emulsion polymerization process, the prepared boron affinity-assisted acetylglucosamine surface imprinted material is in a standard spherical shape, the particle size distribution is counted, and the particle size is 5.6 +/-1.6 microns.
Example 5
MIP3 recognition of acetylglucosamine and acetylglucosamine modified peptide fragments
The adsorption performance of MIP3 and NIP3 on acetylglucosamine was evaluated using 80% ACN as an incubation solvent, as shown in fig. 6. It can be seen that the adsorption capacity of MIP3 to acetylglucosamine is higher than that of NIP3, which indicates that MIP3 has higher affinity to acetylglucosamine due to the existence of the imprinted site.
To evaluate selectivity of MIP3 for acetylglucosamine, the adsorption capacity of MIP3 for three small molecules was determined, respectively, using serine (Ser) and glycine (Gly) as references. As shown in FIG. 6, the adsorption capacity of MIP3 on acetylglucosamine is highest, reaching 0.30 mg/g; while the adsorption capacity for Ser and Gly was only 0.12 and 0.04 mg/g. This indicates that MIP3 has better selectivity for acetylglucosamine in a simple small molecule system.
To further evaluate the enrichment performance of MIP3, it was used for enrichment of acetylglucosamine modified peptide fragments in the enzymatic hydrolysate of murine brain tissue. As shown in fig. 8, a total of 12 acetylglucosamine modified proteins were identified from the original rat brain tissue hydrolysate, corresponding to 18 acetylglucosamine modified peptide fragments and 50 PSMs. After MIP3 enrichment, the number of the identified acetylglucosamine modified proteins is increased to 15, the number of the acetylglucosamine modified peptide fragments is increased to 42, and the number of corresponding PSMs is increased to 242, which indicates that MIP3 has better enrichment capacity on the acetylglucosamine modified peptide fragments.
Example 6
The acetylglucosamine imprinted bulk material MIP4 can be obtained by using methacrylic acid as a functional monomer, methylene bisacrylamide as a cross-linking agent and ammonium persulfate/sodium bisulfite as a redox initiator, wherein the mixing mass ratio of the acetylglucosamine, the functional monomer, the cross-linking agent, the initiator and the solvent is 30:40:5:5:20, and the other steps are the same as those in example 1.
Example 7
The acetylglucosamine surface imprinted material MIP5 was obtained by using 10mg of silicon nanoparticles bonded with acetylglucosamine as a stabilizer, vinylbenzene as a functional monomer, and toluene as an oil phase solvent, wherein the mass ratio of the functional monomer, the crosslinking agent, the initiator and the solvent in the oil phase was 11:72:4.15:12.85, and the same as example 2.
Example 8
The matrix material of the stabilizer was dissolved in 4M aqueous sodium hydroxide by treating the polymer 4 times for 6 h/time with 5mL of 4M aqueous sodium hydroxide. Otherwise, as in example 3, the acetylglucosamine surface imprinted material MIP6 was obtained.
Claims (6)
1. An acetylglucosamine imprinted material, which is characterized in that:
the method comprises the steps of (1) taking N-acetylglucosamine-D as a template, carrying out copolymerization on a functional monomer and a cross-linking agent in the presence of template molecules by using a bulk molecular imprinting method or a Pickering emulsion molecular imprinting method, and removing the template molecules to prepare the acetylglucosamine imprinted material; and the material is used for the recognition of the acetylglucosamine modified peptide fragment.
2. The imprinting material of claim 1, wherein:
acetyl glucosamine bulk blotting: mixing the acetylglucosamine, the functional monomer, the cross-linking agent, the initiator and the solvent, polymerizing, and removing template molecules to form the body imprinting material of the acetylglucosamine;
the functional monomer refers to a monomer capable of polymerizing to form a high molecular polymer, and comprises acrylamide, methacrylamide, isopropyl acrylamide, hydroxyethyl acrylamide, phenylpropenamide, acrylic acid, methyl acrylate, butyl acrylate, methacrylic acid, methyl methacrylate, ethyl methacrylate, butyl methacrylate, tetramethoxy silane, amino trimethoxy silane and vinyl trimethoxy silane, one or more of butyltrimethoxysilane, trimethoxysilylphenylboronic acid, tetraethoxysilane, aminotriethoxysilane, vinyltriethoxysilane, butyltriethoxysilane, triethoxysilane phenylboronic acid, dopamine, vinylpyrrolidone, vinylimidazole, vinyloxirane, styrene, vinylphenylboronic acid, vinylbenzoic acid, methoxypropene, and allyl methyl ether;
the cross-linking agent comprises one or two of methylene bisacrylamide and ethylene glycol dimethacrylate;
the initiator comprises azo initiator or redox initiator such as ammonium persulfate and one or more of sodium bisulfite, potassium persulfate and sodium bisulfite;
the solvent comprises one or more of water, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, chloroform, tetrahydrofuran, dimethyl sulfoxide, dimethylacetamide and dimethylformamide;
in the bulk imprinting system, the mass fraction of the acetylglucosamine in the total system is 0.1-40%; the mass fraction of the functional monomer in the total system is 5-90%; the mass fraction of the cross-linking agent in the total system is 5-45%; the mass fraction of the solvent in the total system is 0.01-10%; the mass fraction of the initiator in the total system is 0.01-10%;
the template molecule removing method comprises the steps of sequentially eluting the imprinted material by using a C1-C4 alcohol solvent or a C1-C4 alcohol acetic acid solution; the volume concentration of acetic acid in the alcohol acetic acid solution is more than 0-50%.
3. The imprinting material of claim 1, wherein:
acetyl glucosamine pickering emulsion blotting: taking solid particles bonded with acetylglucosamine groups as a stabilizer, adding a mixed solution of a water phase and an oil phase to form a stable emulsion, and removing the stabilizer and a template after initiating polymerization to form the acetylglucosamine imprinted microspheres;
the water phase consists of a water-soluble functional monomer, an aqueous solution and a stabilizer; wherein the functional monomer comprises one or more than two of acrylamide, methacrylamide, isopropyl acrylamide, hydroxyethyl acrylamide, acrylic acid and methacrylic acid, and the mass concentration of the functional monomer is 1-20%; the mass concentration of the stabilizer is 1-15%;
the oil phase is an oil-soluble functional monomer, a cross-linking agent, an initiator and a solvent; the functional monomer comprises one or more than two of vinyl pyrrolidone, vinyl imidazole, vinyl ethylene oxide, styrene, vinyl phenylboronic acid, vinyl benzoic acid, methoxy propylene and allyl methyl ether, the cross-linking agent comprises one or more than two of methylene bisacrylamide and ethylene glycol dimethacrylate, the initiator is an azo initiator comprising one or more than two of azobisisobutyronitrile, azobisisoheptonitrile and azobisisobutylamidine hydrochloride, and the solvent comprises one or more than two of toluene, chloroform, acetone and tetrahydrofuran; the mass fraction of the functional monomer in the total system is 0-30%; the mass fraction of the cross-linking agent in the total system is 10-98%; the mass fraction of the initiator in the total system is 0.01-10%; the mass fraction of the solvent in the total system is 0-50%; the volume ratio of the oil phase in the oil-water phase is 1-90%.
4. The imprinting material of claim 3, wherein:
the solid particles bonded with the acetylglucosamine groups are used as a stabilizer, and the acetylglucosamine is bonded on the surface of a matrix material by utilizing covalent bonds, so that the solid particles are used as the stabilizer; the matrix material comprises one or more than two of silicon matrix and metal oxide matrix; the acetyl glucosamine bonding process is to form a covalent bond between hydroxyl on the acetyl glucosamine and a matrix material; or introducing other reactive groups on the acetylglucosamine, wherein the reactive groups comprise one or more than two of alkynyl, azide, alkenyl, sulfydryl, amino, carboxyl and epoxy groups, so that the acetylglucosamine is bonded on the matrix material.
5. The imprinting material of claim 3 or 4, wherein:
the method for removing the stabilizer comprises the following steps: dissolving a silicon substrate and a metal oxide substrate by using strong acid and strong base; the strong acid comprises one or more than two of hydrofluoric acid, ammonium hydrofluoride, hydrochloric acid and sulfuric acid with the concentration of 500mM-5M, and the strong base comprises one or more than two of sodium hydroxide, potassium hydroxide, barium hydroxide and calcium hydroxide with the concentration of 500 mM-5M;
the template molecule removing method comprises the steps of sequentially eluting the imprinted material by using a C1-C4 alcohol solvent or a C1-C4 alcohol acetic acid solution; the volume concentration of acetic acid in the alcohol acetic acid solution is 0-50%.
6. Use of an acetylglucosamine imprinted material according to any one of claims 1 to 5 for the recognition of an acetylglucosamine-modified peptide fragment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711035056.0A CN109721759B (en) | 2017-10-30 | 2017-10-30 | Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711035056.0A CN109721759B (en) | 2017-10-30 | 2017-10-30 | Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109721759A CN109721759A (en) | 2019-05-07 |
| CN109721759B true CN109721759B (en) | 2021-08-03 |
Family
ID=66291480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711035056.0A Active CN109721759B (en) | 2017-10-30 | 2017-10-30 | Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109721759B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110204711B (en) * | 2019-05-31 | 2021-11-30 | 中国药科大学 | Preparation method and application of pomegranate-like structure hollow mesoporous molecularly imprinted polydopamine nanoparticle adsorbent |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103406109A (en) * | 2013-08-06 | 2013-11-27 | 南京大学 | Controllable and universal directional surface printing method and application of molecular printing polymer obtained via same |
| CN104069836A (en) * | 2013-03-29 | 2014-10-01 | 中国科学院大连化学物理研究所 | Molecularly imprinted microsphere material based on polymer phase inversion self assembly and application thereof |
| CN104174390A (en) * | 2014-08-20 | 2014-12-03 | 河南科技大学 | Preparation method and application of ethopabate molecular imprinting solid phase extraction small column |
| CN104941602A (en) * | 2014-03-27 | 2015-09-30 | 中国科学院大连化学物理研究所 | Protein antigen determinant molecularly imprinted material as well as preparation and application thereof |
-
2017
- 2017-10-30 CN CN201711035056.0A patent/CN109721759B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104069836A (en) * | 2013-03-29 | 2014-10-01 | 中国科学院大连化学物理研究所 | Molecularly imprinted microsphere material based on polymer phase inversion self assembly and application thereof |
| CN103406109A (en) * | 2013-08-06 | 2013-11-27 | 南京大学 | Controllable and universal directional surface printing method and application of molecular printing polymer obtained via same |
| CN104941602A (en) * | 2014-03-27 | 2015-09-30 | 中国科学院大连化学物理研究所 | Protein antigen determinant molecularly imprinted material as well as preparation and application thereof |
| CN104174390A (en) * | 2014-08-20 | 2014-12-03 | 河南科技大学 | Preparation method and application of ethopabate molecular imprinting solid phase extraction small column |
Non-Patent Citations (3)
| Title |
|---|
| "Immunostimulating peptide interfacial imprinted magnetic microspheres synthesized via Pickering emulsion polymerization";Xiaoyan Zhang et al.;《Journal of Material Science》;20161227;第52卷(第8期);摘要，第4714页右栏第3段至第4716页右栏末段，机理1，图1 * |
| "Molecular Imprinting Techniques Used for the Preparation of Biosensors";Gizem Ertürk et al.;《sensors》;20170204;第17卷(第2期);1-17 * |
| "Ultratrace Enrichment of Tyrosine Phosphorylated Peptides on an Imprinted Polymer";Stefan Helling et al.;《Anal. Chem.》;20110209;第83卷(第5期);摘要，第1862页右栏末段，supporting information第4页末段至第5页首段，第1864页右栏末段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109721759A (en) | 2019-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Recent advances and trends of molecularly imprinted polymers for specific recognition in aqueous matrix: Preparation and application in sample pretreatment | |
| Hajizadeh et al. | Cryogelation of molecularly imprinted nanoparticles: A macroporous structure as affinity chromatography column for removal of β-blockers from complex samples | |
| CN110115992B (en) | Aptamer functionalized polymer column for specific recognition of mycotoxin and preparation method thereof | |
| Zheng et al. | Specific enrichment of glycoproteins with polymer monolith functionalized with glycocluster grafted β-cyclodextrin | |
| JPS5858026B2 (en) | Packing material for chromatography and its manufacturing method | |
| Zhu et al. | Recent development of monolithic materials as matrices in microcolumn separation systems | |
| CN1815224A (en) | Capillary liquid-phase chromatographic collumn and making method | |
| CN112111066A (en) | Preparation method and application of core-shell structure-based polymer microspheres for protein separation and analysis | |
| CN113274993A (en) | Preparation method of silica gel matrix chromatographic packing for separating strong-polarity drugs | |
| CN109721759B (en) | Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment | |
| Ying et al. | Preparation and specific recognition of protein macromolecularly imprinted polyampholyte hydrogel | |
| Zhang et al. | Deep eutectic solvents-based polymer monolith incorporated with titanium dioxide nanotubes for specific recognition of proteins | |
| CN103289030A (en) | Novel method for efficiently grafting glycidyl methacrylate (GMA) on surface of silica gel | |
| CN109400823B (en) | Octavinyl-POSS and ethylene glycol dimethacrylate co-crosslinked boron affinity monolithic column and preparation method thereof | |
| CN101464439B (en) | Production method for protein molecule imprinting integral column | |
| CN112337441B (en) | Phenylboronic acid-chitosan modified monolithic column and preparation method and application thereof | |
| CN109342626A (en) | POSS organic-inorganic hybrid molecule trace integral post of specific recognition ochratoxin A and preparation method thereof | |
| Wang et al. | Graft copolymerization of anion and cation onto silica and application in mixed-mode of reversed phase/hydrophilic interaction/ion exchange chromatography | |
| CN115926046A (en) | Catalpol magnetic surface molecularly imprinted polymer and preparation method and application thereof | |
| CN105237770A (en) | Preparation method of molecularly imprinted polymer | |
| Wang et al. | Dual-function monolithic enzyme reactor based on dopamine/graphene oxide coating for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment | |
| Li et al. | Preparation and evaluation of uniform-size (-)-ephedrine-imprinted polymeric microspheres by multi-step swelling and suspension polymerization | |
| Ding et al. | Preparation of pH‐responsive metal chelate affinity polymer for adsorption and desorption of insulin | |
| Song et al. | Molecularly imprinted monoliths: Recent advances in the selective recognition of biomacromolecules related biomarkers | |
| Yu et al. | RECENT DEVELOPMENT AND APPLICATION OF MONOLITHIC COLUMNS. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |