CN104403043A - Molecularly imprinted microsphere for separated histone H4-K16 acetylation labelling polypeptide - Google Patents

Abstract

A method for preparing molecularly imprinted microspheres for isolating histone H4-K16 acetylation-marked polypeptides, comprising the following steps: first grafting bifunctional glutaraldehyde on the surface of silica gel, then bonding imprinted molecules on the surface of silica gel, and finally synthesizing molecularly imprinted microspheres polymer microspheres. The advantage of the present invention is: the molecularly imprinted polymer microspheres have good recognition ability and selectivity for the labeled peptide GGAKacR after hydrolysis of histone H4 containing acetylated K16, and can be used for the hydrolysis of histone H4 samples containing acetyl Selective enrichment and determination of the labeled peptide of Fak16; at the same time, because the imprinting site is on the surface of the material, the molecularly imprinted polymer microspheres can also selectively extract long-chain peptides with the GGAKacR sequence at the C-terminus. In the extraction experiment of histone hydrolysis samples, the recovery rate of the molecularly imprinted microspheres for peptides reached over 80%, which can play a role in the determination of histone H4-K16 acetylation.

Description

Molecularly imprinted microspheres for isolation of histone H4-K16 acetylation-tagged peptides

technical field

The invention relates to the preparation of molecularly imprinted materials, in particular to a molecularly imprinted microsphere for separating histone H4-K16 acetylation marked polypeptide and a preparation method thereof.

Background technique

Histone is a basic protein in the chromatin of eukaryotic cells, which plays an important role in chromosome assembly. It has been found that histone has a variety of post-translational modifications, and these post-translational modifications play an important role in regulating the dynamic expression of genes. significance. Histone H4 (Histone H4) is one of the four core histones (H2A, H2B, H3, H4). Studies have found that the sixteenth lysine at the N-terminal of histone H4 (referred to as H4-K16) Acetylation modification plays an important role in the assembly of chromosomes. This modification is not only related to chromatin folding defects, but also affects cell cycle regulation. Therefore, the analysis of histone H4-K16 acetylation is of great significance. Since protein post-translational modifications are often in dynamic changes and their abundance is low, their analysis and identification are difficult. Therefore, it is necessary to use selective enrichment materials for extraction and separation. On the other hand, selective biological antibodies There are problems of high price and poor environmental tolerance. Therefore, it is very meaningful to prepare artificial synthetic materials that can selectively enrich the labeled peptides of histone H4-K16 acetylation.

Molecular imprinting (molecular imprinting) is a technology for preparing polymers with recognition ability, and non-covalent molecular imprinting is a commonly used method for imprinting technology. In non-covalent molecular imprinting, template molecules (also known as imprinted molecules) interact with functional monomers to form complexes, and polymerize in the presence of cross-linking agents to form imprinted polymers, which are obtained after elution to remove imprinted molecules in the material Molecularly imprinted polymer (molecularly imprinted polymer, abbreviated as MIP). There are cavities in the MIP that match the size and functional groups of the imprinted compounds, and these cavities form the recognition sites for the imprinted molecules. Since the recognition ability of MIP is similar to that of biological antibodies, molecular imprinting technology is also known as a technology for preparing artificial antibodies. In the synthesis of macromolecular imprinting materials such as proteins, in order to reduce mass transfer resistance, the surface-confined imprinting method (Surface-confinedimprinting) is applied to molecular imprinting. Afterwards, the immobilization medium is removed to obtain the imprinted material with binding sites limited to the surface. This imprinted polymer has an easily accessible binding site and can bind proteins or polypeptides that contain imprinted molecular segments and are larger than imprinted molecules, which has strong advantages in Western blotting. Partial peptides in protein molecules that can bind to antibodies are selected as imprinted molecules for imprinting, and synthetic MIP is used for the binding of the entire protein, which is called epitope imprinting.

The present invention synthesized a molecularly imprinted polymer microsphere capable of recognizing histone H4-K16 acetylation (marked as H4-K16ac), because the hydrolyzed histone H4 peptide (GGAKacR) containing acetylated K16 can be used as The marker of H4-K16ac, GGAKacR is used as the imprinted template molecule in the synthesis, and it is immobilized on the surface of silica gel; trifluoromethacrylic acid is used as the functional monomer, and N'N-vinylbisacrylamide is used as the crosslinking agent. Molecularly imprinted synthesis was carried out on the surface of imprinted molecule-imprinted silica gel; GGAKacR immobilized silica gel was removed after synthesis to obtain GGAKacR molecularly imprinted microspheres (marked as GGAKacR-MIP).

Contents of the invention

The purpose of the present invention is to provide a polypeptide molecularly imprinted microsphere and its preparation method in view of the above-mentioned technical analysis and existing problems. The peptide (GGAKacR) has good recognition ability and selectivity, and can be used for the selective enrichment and determination of labeled peptides containing acetylated K16 in histone H4 hydrolysis samples.

Technical scheme of the present invention:

A molecularly imprinted microsphere for separating histone H4-K16 acetylation-marked polypeptides, which is a cross-linked polymer with trifluoromethacrylic acid as a monomer and N'N-vinylbisacrylamide as a cross-linking agent , the particle size is about 20 microns, and the surface of the polymer has a binding site that recognizes GGAKacR and a long-chain polypeptide containing a GGAKacR peptide at the C-terminus; the microsphere material has good selectivity for GGAKacR, as an adsorption material, molecularly imprinted polymerization The adsorption capacity of the microspheres for GGAKacR in phosphate buffer is about 50 μmol·g ^-1 ; the imprinting factor of the microsphere material for imprinted molecules is 2.5, and the imprinting factor is calculated by IF=Q _MIP /Q _NIP , where Q _MIP is The adsorption capacity of molecularly imprinted polymers for imprinted molecules (μmol·g ^-1 ), Q _NIP is the adsorption capacity of non-imprinted polymers for imprinted molecules (μmol·g ^-1 ); molecularly imprinted polymer microspheres were used as chromatographic stationary phase , with phosphate buffer (20mM, pH=7.0) as the mobile phase, the retention factor of the imprinted molecule GGAKacR is about 5.9, and the selectivity factor of the molecularly imprinted material determined by liquid chromatography is α≥1.7, and the selectivity factor is determined by α =k _I /k _X calculation, where k _I is the retention factor of the imprinted molecule and k _X is the retention factor of its analog.

A method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides, comprising the following steps:

1. Bifunctional glutaraldehyde grafted on the surface of silica gel;

1) Mix silica gel microspheres with hydrochloric acid solution, reflux at 120°C for 7 hours, filter and wash, wash with distilled water until neutral, then wash once with acetone, and finally dry in vacuum at 90°C for 12 hours to obtain activated silica gel;

2) Mix the above-mentioned activated silica gel with dry toluene, then add 3-aminopropyltriethoxysilane, reflux for 12 hours under the protection of nitrogen, wash with toluene, acetone, distilled water, acetone in turn after the reaction is completed, and finally in 60 ℃ Dry under vacuum for 12 hours to obtain aminopropyl bonded silica gel (Sil-NH ₂ );

3) Add aminopropyl bonded silica gel (Sil-NH ₂ ) and glutaraldehyde solution into phosphate buffer solution and mix well, react at 30°C for 6 hours, filter with suction after the reaction is completed, and wash with distilled water until completely removed Unreacted glutaraldehyde was washed once with acetone, and the obtained microspheres were vacuum-dried at 60°C for 12 hours to obtain aldehyde-bonded silica gel (Sil-CHO);

2. Bond imprinted molecules on the surface of silica gel

1) Dissolve the polypeptide (GGAKacRpbf) in the newly prepared phosphate buffer solution, then add aldehyde-bonded silica gel (Sil-CHO), shake in a shaker at 25°C for 24h, wash with buffer solution and distilled water after the reaction is completed Wash each time once, and after vacuum drying, the peptide-immobilized silica gel is obtained;

2) Mix the above peptide-immobilized silica gel with trifluoroacetic acid (TFA) solution and stir at room temperature to remove the protective group of arginine, filter through a sand core funnel, rinse with distilled water until neutral, and finally dehydrated ethanol After cleaning, vacuum-dry at 60°C for later use to obtain GGAKacR-supported silica gel (Sil-GGAKacR);

3. Synthesis of Molecularly Imprinted Polymer Microspheres

1) Dissolving functional monomers, cross-linking agents and initiators in phosphate buffer to configure a pre-polymerization solution;

2) Immerse the GGAKacR immobilized silica gel (Sil-GGAKacR) in the pre-polymerization solution, let it stand at room temperature for 3-4 hours, take it out, rinse it with ethanol quickly, transfer it to a conical container, pass nitrogen gas to remove oxygen, seal it, and place it in an oven React at 45°C for 24h, transfer the obtained microspheres into a conical container, add 10mL of acetone, and then add 1.5mL of 40wt% HF solution, place it in an ice bath for 1h, then shake at room temperature for 12h, and obtain the dissolved silica gel. imprinted polymer;

3) The imprinted polymer after dissolving the silica gel was collected by suction filtration, washed with acetone and distilled water in turn until neutral, then dried, then Soxhlet extracted with acetonitrile-acetic acid-water mixed solution, then extracted with methanol, and vacuum-dried The target product molecularly imprinted polymer microspheres (GGAKacR-MIP) was obtained.

The particle diameter of described silica gel microsphere is 10-80 micron, and aperture is The volume ratio of distilled water to hydrochloric acid in the hydrochloric acid solution used for silica gel activation is 1:1; the dosage ratio of silica gel microspheres to hydrochloric acid solution is 0.1g:1mL.

In the reaction of activated silica gel and 3-aminopropyltriethoxysilane, the dosage ratio of activated silica gel, toluene and 3-aminopropyltriethoxysilane is 4.5g:45mL:5.3mL.

The concentration of the glutaraldehyde solution is 2.6mol/L; the concentration of the phosphate buffer solution is 20mmol, and the pH is 7.0; the consumption ratio of aminopropyl bonded silica gel, glutaraldehyde solution and phosphate buffer solution is 4.5g: 2.0mL: 25mL.

The concentration of the phosphate buffer solution is 20 mmol, and the pH is 7.0; the dosage ratio of the polypeptide, the phosphate buffer solution and the aldehyde-bonded silica gel is 80.0 mg: 20 mL: 2.0 g.

The concentration of the TFA solution is 80v%, and the dosage ratio of the peptide-immobilized silica gel to the TFA solution is 2.0g:15mL.

The functional monomer is trifluoromethacrylic acid, methacrylic acid or acrylamide; the crosslinking agent is N'N-vinylbisacrylamide or N'N-methylenebisacrylamide; the initiator is persulfuric acid Ammonium; the concentration of the phosphate buffer solution is 20mmol, and the pH is 7.0; when using N'N-ethylenebisacrylamide as the cross-linking agent, the amount ratio of the functional monomer, cross-linking agent, initiator and phosphate buffer 126mg: 219mg: 10.3mg: 1.5mL, when N'N-methylenebisacrylamide is used as the crosslinking agent, the dosage ratio of functional monomer, crosslinking agent, initiator and phosphate buffer is 126mg: 30mg : 6.0mg: 1.5mL.

The dosage ratio of the GGAKacR imprinted silica gel, acetone and HF solution is 0.5 g: 10 mL: 1.5 mL.

The volume ratio of acetonitrile, acetic acid and distilled water in the acetonitrile-acetic acid-water mixed solution is 5:5:90.

The advantage of the present invention is that the molecularly imprinted polymer microspheres have good recognition ability and selectivity for the hydrolyzed histone H4 peptide (GGAKacR) containing acetylated K16, and can be used in the hydrolyzed histone H4 samples containing Selective enrichment and determination of acetylated K16-labeled peptides; at the same time, because the imprinting site is on the surface of the material, the molecularly imprinted polymer microspheres can also selectively extract long-chain peptides containing the GGAKacR sequence at the C-terminus. In the extraction experiment of spiked histone hydrolyzed samples, the recovery rate of the molecularly imprinted microspheres for GGAKacR reached more than 80%, which can play a role in the determination of histone H4-K16 acetylation.

Description of drawings

Figure 1 is a scanning electron microscope image of imprinted polymer microspheres.

Figure 2 is the equilibrium adsorption isotherms of the imprinted polymer microspheres and non-imprinted microspheres for the imprinted molecule GGAKacR.

Fig. 3 is a comparison chart of the adsorption amount of the imprinted polymer microspheres to the imprinted molecule GGAKacR and its analogues.

Fig. 4 is a liquid chromatography separation diagram of imprinted polymer microspheres to imprinted molecule GGAKacR and its analogues.

Figure 5 is the mass spectrogram of the spiked histone hydrolyzate and the eluate after adsorption by imprinted polymer microspheres.

Figure 6 is a liquid chromatogram of the spiked histone enzymatic hydrolyzate and the eluate after being absorbed by the imprinted polymer microspheres, in which: H4-K12/16Ac represents GLGKacGGAKacR.

Detailed ways

The reagents used in the following examples are all analytically pure; the medicines are standard or reference substances.

Example:

A molecularly imprinted microsphere for separating histone H4-K16 acetylation-marked polypeptides, which is a cross-linked polymer with trifluoromethacrylic acid as a monomer and N'N-vinylbisacrylamide as a cross-linking agent , the particle size is 20 microns, and the surface of the polymer has a binding site that recognizes GGAKacR and a long-chain polypeptide containing a GGAKacR peptide at the C-terminus; the microsphere material has good selectivity for GGAKacR, and as an adsorption material, molecularly imprinted polymer The adsorption capacity of microspheres for GGAKacR in phosphate buffer is about 50 μmol g ^-1 ; the imprinting factor of microsphere materials for imprinted molecules is 2.5, and the imprinting factor is calculated by IF=Q _MIP /Q _NIP , where Q _MIP is The saturated adsorption capacity of molecularly imprinted polymers for imprinted molecules (μmol·g ^-1 ), Q _NIP is the saturated adsorption capacity of non-imprinted polymers for imprinted molecules (μmol·g ^-1 ); molecularly imprinted polymer microspheres were used as chromatographic The stationary phase is a phosphate buffer with a concentration of 20mM and pH=7.0 as the mobile phase. The retention factor of the imprinted molecule GGAKacR is 5.9. The selectivity factor α≥1.7 of the molecularly imprinted material is determined by liquid chromatography, and the selectivity factor is determined by α =k _I /k _X calculation, where k _I is the retention factor of the imprinted molecule and k _X is the retention factor of its analog.

A method for preparing molecularly imprinted polymer microspheres for identifying and separating histone H4-K16 acetylation-marked polypeptides, comprising the following steps:

1. Grafting bifunctional glutaraldehyde on the surface of silica gel

1) the particle size is 20 microns, the average pore size is Add 4.5 g of silica gel microspheres with a specific surface area of 232 m ² /g into a 100 mL round bottom flask, add hydrochloric acid aqueous solution (hydrochloric acid/water volume ratio is 1:1), reflux at 120°C for 7 hours, filter and wash, and then wash with distilled water until Neutral, then washed once with acetone, and finally vacuum-dried at 90°C for 12 hours to obtain activated silica gel;

2) Mix 4.5g of the above-mentioned activated silica gel with 45mL of dry toluene, then add 5.3mL of 3-aminopropyltriethoxysilane, and reflux for 12h under nitrogen protection. After the reaction is completed, wash with toluene, acetone, distilled water, and acetone in sequence , and finally vacuum-dried at 60°C for 12 hours to obtain aminopropyl bonded silica gel (Sil-NH ₂ ). Calculated by the increase of nitrogen element content in elemental analysis, the graft density of aminopropyl group is 5.1 μmol·m ^-2 ;

3) Add 4.5 g of aminopropyl-bonded silica gel (Sil-NH ₂ ) and 2.0 mL of glutaraldehyde solution with a concentration of 2.6 mol/L into 25 mL of newly prepared 20 mM phosphate buffer solution with pH 7.0 and mix Evenly, in a water bath at 30°C for 6 hours, after the reaction is completed, suction filter, first wash with distilled water to completely remove unreacted glutaraldehyde, then wash with acetone once, and dry the rust red microspheres at 60°C for 12 hours in vacuum to obtain Aldehyde-bonded silica gel (Sil-CHO), calculated through the increase of carbon element content in elemental analysis, the grafting density of aldehyde group is 3.6μmol·m ^-2 ;

2. Bond imprinted molecules on the surface of silica gel

1) Dissolve 80.0 mg of a polypeptide with a protective group (GGAKacRpbf) in 20 mL of newly prepared 20 mM, pH 7.0 phosphate buffer solution, then add 2.0 g of aldehyde-bonded silica gel (Sil-CHO), and shake Shake at 25°C for 24 hours, wash with buffer solution and distilled water once after the reaction is completed, and dry in vacuum to obtain peptide-immobilized silica gel;

2) Mix the above 2.0 g of polypeptide-supported silica gel with 15 mL, 80 vt% trifluoroacetic acid (TFA) solution and stir at room temperature to remove the protective group of arginine, filter through a sand core funnel, and rinse with distilled water until Neutral, finally washed with absolute ethanol and dried in vacuum at 60°C for later use to obtain GGAKacR-supported silica gel (Sil-GGAKacR);

3. Synthesis of Molecularly Imprinted Polymer Microspheres

1) Dissolve the functional monomer trifluoromethacrylic acid, cross-linking agent N'N-vinylbisacrylamide and initiator ammonium persulfate in phosphate buffer to prepare a pre-polymerization solution, functional monomer, cross-linking agent The dosage ratio of coupling agent, initiator and phosphate buffer solution is 126mg: 219mg: 10.3mg: 1.5mL;

2) Immerse the GGAKacR immobilized silica gel in the pre-polymerization solution, let it stand at room temperature for 3 hours, take it out, rinse it with ethanol quickly, transfer it to a conical container, and seal it with nitrogen gas to remove oxygen, and put it in an oven for 24 hours at 45°C. , transfer the obtained microspheres to a conical container, add 10mL of acetone, and then add 1.5mL of 40wt% HF solution, place in an ice bath for 1h, then shake at room temperature for 12h, and obtain the imprinted polymer after dissolving silica gel;

3) The imprinted polymer after dissolving the silica gel was collected by suction filtration, washed with acetone and distilled water to neutrality and then dried, and then performed Soxhlet extraction with acetonitrile-acetic acid-water mixed solution with a volume ratio of 5:5:90. The target product molecularly imprinted polymer microspheres (GGAKacR-MIP) was obtained after extraction with methanol and vacuum drying.

Figure 1 is a scanning electron microscope image of the imprinted polymer. In the scanning electron microscope image, the imprinted polymer is spherical, with a particle size of about 20 microns, which is similar to silica gel, indicating that the polymerization using silica gel as a sacrificial medium was successful.

The synthesis of non-imprinted polymer microspheres (NIP) was the same as the synthesis of imprinted polymer microspheres except that aldehyde-bonded silica gel (Sil-CHO) was used instead of GGAKacR-supported silica gel.

4. Evaluation of Molecularly Imprinted Polymer Microspheres (GGAKacR-MIP):

1) Evaluation of the affinity of molecularly imprinted polymer GGAKacR-MIP by equilibrium adsorption method

The adsorption capacity of the imprinted polymer for the imprinted polypeptide GGAKacR was determined by equilibrium adsorption method. By comparing the adsorption capacity (Q) of imprinted and non-imprinted polymers for GGAKacR and calculating the imprinting factor (IF), the affinity and imprinting effect of imprinted polymer materials for imprinted polypeptides and polypeptides similar in structure to them were evaluated.

Weigh 10 parts of 5 mg of GGAKacR-MIP obtained in the examples and place them in adsorption bottles, add GGAKacR solutions with different concentrations (0.1–2.0 mmol·L ^-1 ) respectively, and the solution is filled with Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution ( 20mM, pH 7.0) was prepared as a solvent, the adsorption mixture was placed in a water bath shaker, and oscillated and adsorbed at room temperature for 24h. Centrifuge and separate the supernatant for liquid chromatography detection. The liquid chromatography conditions are: chromatographic column: Phenomenex C18, 10 μm, 250×4.6mm, mobile phase: acetonitrile/water (7/93, volume ratio), containing 0.1% TFA aqueous solution, flow rate: 1.0mL/min, detection wavelength: 205nm. The adsorption capacity of the polymer for the polypeptide is calculated by Q=(C ₀ -C)V/W, where Q: adsorption capacity; C ₀ : the concentration of the polypeptide in the solution before adsorption; C: the concentration of the polypeptide in the solution after adsorption; W : the mass of GGAKacR-MIP; V: the volume of the adsorption solution added.

Figure 2 shows the equilibrium adsorption isotherms of the imprinted and non-imprinted polymer microspheres for the imprinted molecule GGAKacR. The results show that the adsorption amount of the imprinted polymer for GGAKacR is significantly higher than that of the non-imprinted polymer, and the saturated adsorption amount of GGAKacR-MIP for GGAKacR It is 2.5 times that of the non-imprinted polymer (NIP), that is, the imprinting factor IF=2.5, indicating that the binding of the imprinted polymer comes from the imprinting effect.

2) Evaluation of the selectivity of molecularly imprinted polymer GGAKacR-MIP by equilibrium adsorption method

GGVKacR, GGAKR, GGAK, GLGKacGGAKacR, and GGKacGLGKacGGAKacR were selected as structural analogues of the imprinted molecule GGAKacR for selectivity evaluation. Adsorption solutions were prepared with GGAKacR and its structural analogues respectively [concentration of 1.0 mmol/L, Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer (20 mM, pH 7.0) as solvent]. Weigh 5 mg of the GGAKacR-MIP (or NIP) obtained in Example 3 and place them in adsorption bottles, add 2.0 mL of different polypeptide adsorption solutions, seal them, place them in a water bath shaker, and oscillate and adsorb at room temperature for 24 hours. After the adsorption was completed, centrifuged, the supernatant was taken for liquid chromatography detection,

The liquid chromatography conditions are: chromatographic column: Phenomenex C18, 10 μm, 250×4.6mm, mobile phase A: aqueous solution containing 0.1% TFA, mobile phase B: acetonitrile solution containing 0.1% TFA. Gradient elution: 0-6min, 1-7% B; 6-8min, 7% B; 8-15min, 7-15% B; 15-25min, 15-35% B; flow rate: 1.0mL/min, detection Wavelength: 205nm.

The adsorption capacity of GGAKacR-MIP for various polypeptides is calculated by Q=(C ₀ -C)V/W, where Q: adsorption capacity; C ₀ : the concentration of polypeptides in the solution before adsorption; C: the concentration of polypeptides in the solution after adsorption Concentration; W: the quality of GGAKacR-MIP; V: the volume added by the adsorption solution. The adsorption amount of polypeptide on NIP was determined in the same way. The imprinting selectivity of GGAKacR-MIP is calculated by IS=Q _MIP /Q _NIP , where Q _MIP is the adsorption amount of polypeptide on MIP (μmol·g ^-1 ), and Q _NIP is the adsorption amount of polypeptide on NIP (μmol·g ^-1 ). Fig. 3 is a graph comparing the adsorption capacities of the imprinted polymers for the equilibrium adsorption of the imprinted molecule GGAKacR and its analogues. The figure shows: Comparing the adsorption capacity of GGAKacR-MIP and NIP for imprinted molecules and analogues (Figure 3), it can be seen that the imprinted material has the highest adsorption capacity and imprinted selectivity for imprinted molecules (GGAKacR), followed by GGAKacR at the C-terminus. The long-chain polypeptides of peptides (GLGKacGGAKacR and GGKacGLGKacGGAKacR). For other analogs (GGVKacR, GGAKR, GGAK), the adsorption capacity and imprinting selectivity are low.

3) Determination of the selectivity of GGAKacR-MIP by liquid chromatography

Using GGAKacR-MIP and NIP as chromatographic stationary phases, the properties of imprinted polymers were determined by chromatography.

The molecularly imprinted polymer microspheres GGAKacR-MIP and the non-molecularly imprinted polymer microspheres NIP synthesized in the examples were packed into stainless steel chromatographic columns with specifications of 20×3.0mm under the vacuum condition and wet packing method. In , after equilibrating the column with phosphate buffer (pH 7.0), the retention of GGAKacR and its analogues on the column was tested respectively.

Fig. 4 is a liquid chromatographic separation diagram of the imprinted polymer to the imprinted molecule GGAKacR and its analogues.

The liquid chromatography conditions are: mobile phase: phosphate buffer (20 mM, pH=7.0), flow rate: 0.1 mL/min, detection wavelength: 205 nm. The samples are GGAKacR, GGVKacR, GGAKR, GGAK, GLGKacGGAKacR, GGKacGLGKacGGAKacR.

The retention factor and selectivity of GGAKacR-MIP for each polypeptide are listed in Table 1. The retention factor is calculated by k=(t _r -t ₀ )/t ₀ , where t _r is the retention time of the analyte, and t ₀ is dead time (measured with acetone). The selectivity factor is calculated by α = k _I /k _X , where k _I is the retention factor of the imprinted molecule and k _X is the retention factor of its analogue. By comparing the retention factors of GGAKacR and similar peptides, it can be seen that GGAKacR-MIP has good selectivity for the imprinted molecule GGAKacR and GLGKacGGAKacR and GGKacGLGKacGGAKacR containing GGAKacR peptides.

Table 1 Retention factor and selectivity of molecularly imprinted polymers for GGAKacR and its analog polypeptides

Note: H4-K12/16Ac stands for GLGKacGGAKacR, H4-K5/8/12/16Ac stands for GGKacGLGKacGGAKacR

The results proved that on the GGAKacR-MIP column, the retention of the template polypeptide GGAKacR was the strongest, followed by the long-chain polypeptides containing the same GGAKacR peptide: GLGKacGGAKacR (represented by H4-K12/16Ac) and GGKacGLGKacGGAKacR (represented by H4-K5/8/ 12/16Ac), the GGAKacR-MIP chromatographic column has a separation factor α≥1.7 for imprinted molecules and their analogs. The influence of the composition of the mobile phase on the retention of GGAKacR-MIP was investigated, and the study proved that GGAKacR had the best retention in the imprinting polymerization solvent (phosphate buffer solution).

5. GGAKacR-MIP imprinted polymer is applied to the separation and enrichment of histone hydrolysis peptides containing acetylated K16 in the peptide mixture

GGAKacR and GLGKacGGAKacR (GGAKacR/GLGKacGGAKacR/histone: 1/1/5, molar ratio) were added to the histone hydrolysis solution, and GGAKacR-MIP imprinted polymer microspheres were added for adsorption. The liquid was removed and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and high-performance liquid chromatography (HPLC). The results proved that GGAKacR-MIP had a good selective enrichment effect.

Weigh 1 mg of histone and dissolve it with 0.8 mL of ammonium bicarbonate solution (pH 8.5). Then 200 μL of 0.1 mg·mL ⁻¹ trypsin hydrochloric acid solution was added thereto, and the mass ratio of trypsin to histone was 1/50. The resulting mixture was shaken and reacted overnight at 37°C. Add 5 μL of glacial acetic acid to terminate the enzymatic hydrolysis reaction. Take 400 μL of histone hydrolyzate and mix with 10 μL of the solution (1.0 mmol/L) containing GGAKacR and GLGKacGGAKacR and dilute to 2.0 mL with phosphate buffer. Add 2.0 mL of the histone hydrolysis solution spiked with GGAKacR and GLGKacGGAKacR into the adsorption bottle containing 10 mg of the GGAKacR-MIP synthesized in the example, and oscillate and adsorb for 12 hours. After centrifugation, the supernatant was removed, and the GGAKacR-MIP was washed three times with 500 μL of phosphate solution. Finally, the GGAKacR adsorbed on the imprinted microspheres was eluted twice with glacial acetic acid/acetonitrile/water (5/5/90, v/v), and the eluents were combined, freeze-dried, and re-dissolved. The solution before adsorption and the eluate after adsorption were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and high-performance liquid chromatography (HPLC).

Figure 5 is the mass spectrum of the spiked histone hydrolyzate before and after molecularly imprinted polymer extraction; Figure 6 is the liquid chromatogram of the spiked histone hydrolyzate before and after molecularly imprinted polymer extraction, in the figure: H4-K12/16Ac stands for GLGKacGGAKacR. The results showed that after GGAKacR-MIP adsorption, GGAKacR and GLGKacGGAKacR became the main components of the solution, other components were greatly reduced, and the sample was purified. The recovery rates of GGAKacR and GLGKacGGAKacR were both above 80%. It can be seen that the imprinted material has good selectivity for imprinted molecules and GLGKacGGAKacR, and can play a role in selective separation and enrichment.

Claims (10)

1. A molecularly imprinted microsphere for separating histone H4-K16 acetylation-marked polypeptide, characterized in that: trifluoromethacrylic acid is used as a monomer and N'N-vinylbisacrylamide is used as a crosslinking agent Cross-linked high molecular polymer with a particle size of 20 microns. The surface of the polymer has a binding site for recognizing GGAKacR and a long-chain polypeptide containing a GGAKacR peptide at the C-terminus; the microsphere material has good selectivity for GGAKacR. Materials, molecularly imprinted polymer microspheres have an adsorption capacity of about 50 μmol g ^-1 for GGAKacR in phosphate buffer; the imprinting factor of the microsphere material for imprinted molecules is ≥ 2.5, and the imprinting factor is calculated by IF=Q _MIP /Q _NIP , where Q _MIP is the adsorption capacity of imprinted polymers (μmol·g ^-1 ), Q _NIP is the adsorption capacity of non-imprinted polymers for imprinted molecules (μmol·g ^-1 ); The ball is used as the chromatographic stationary phase, with phosphate buffer (20mM, pH=7.0) as the mobile phase, the retention factor of the imprinted molecule GGAKacR is 5.9, and the selectivity factor α of the molecularly imprinted material determined by liquid chromatography is ≥ 1.7. The property factor is calculated by α= _kI / _kX , where _ki is the retention factor of the imprinted molecule and _kX is the retention factor of its analog. 2. A method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 1, characterized in that it comprises the steps of: 1. Bifunctional glutaraldehyde grafted on the surface of silica gel; 1) Mix silica gel microspheres with hydrochloric acid solution, reflux at 120°C for 7 hours, filter and wash, wash with distilled water until neutral, then wash once with acetone, and finally dry in vacuum at 90°C for 12 hours to obtain activated silica gel; 2) Mix the above-mentioned activated silica gel with dry toluene, then add 3-aminopropyltriethoxysilane, reflux for 12 hours under the protection of nitrogen, wash with toluene, acetone, distilled water, acetone in sequence after the reaction is completed, and finally in 60 ℃ Dry under vacuum for 12 hours to obtain aminopropyl bonded silica gel (Sil-NH ₂ ); 3) Add aminopropyl bonded silica gel (Sil-NH ₂ ) and glutaraldehyde solution into phosphate buffer solution and mix well, react at 30°C for 6 hours, filter with suction after the reaction is completed, and wash with distilled water until completely removed The unreacted glutaraldehyde was washed once with acetone, and the obtained rust-red microspheres were vacuum-dried at 60°C for 12 hours to obtain aldehyde-bonded silica gel (Sil-CHO); 2. Bond imprinted molecules on the surface of silica gel 1) Dissolve the polypeptide (GGAKacRpbf) in the newly prepared phosphate buffer solution, then add aldehyde-bonded silica gel (Sil-CHO), shake in a shaker at 25°C for 24h, wash with buffer solution and distilled water after the reaction is completed Wash each time once, and after vacuum drying, the peptide-immobilized silica gel is obtained; 2) Mix the above peptide-immobilized silica gel with trifluoroacetic acid (TFA) solution and stir at room temperature to remove the protective group of arginine, filter through a sand core funnel, rinse with distilled water until neutral, and finally dehydrated ethanol After cleaning, vacuum-dry at 60°C for later use to obtain GGAKacR-supported silica gel (Sil-GGAKacR); 3. Synthesis of Molecularly Imprinted Polymer Microspheres 1) Dissolving functional monomers, cross-linking agents and initiators in phosphate buffer to configure a pre-polymerization solution; 2) Immerse the GGAKacR immobilized silica gel (Sil-GGAKacR) in the pre-polymerization solution, let it stand at room temperature for 3-4 hours, take it out, rinse it with ethanol quickly, transfer it to a conical container, pass nitrogen gas to remove oxygen, seal it, and place it in an oven React at 45°C for 24h, transfer the obtained microspheres into a conical container, add 10mL of acetone, and then add 1.5mL of 40wt% HF solution, place it in an ice bath for 1h, then shake at room temperature for 12h, and obtain the dissolved silica gel. imprinted polymer; 3) The imprinted polymer after dissolving the silica gel was collected by suction filtration, washed with acetone and distilled water in turn until neutral, then dried, then Soxhlet extracted with acetonitrile-acetic acid-water mixed solution, then extracted with methanol, and vacuum-dried The target product molecularly imprinted polymer microspheres (GGAKacR-MIP) was obtained. 3. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that: the particle diameter of the silica gel microspheres is 10-80 microns, and the pore diameter is The volume ratio of distilled water to hydrochloric acid in hydrochloric acid solution is 1:1; the dosage ratio of silica gel microspheres to hydrochloric acid solution is 0.1g:1mL. 4. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that the amount of silica gel, toluene and 3-aminopropyltriethoxysilane after the activation The ratio is 4.5g:45mL:5.3mL. 5. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that the concentration of the glutaraldehyde solution is 2.6mol/L; the concentration of the phosphate buffer solution 20mmol, pH 7.0; the dosage ratio of aminopropyl bonded silica gel, glutaraldehyde solution and phosphate buffer solution is 4.5g: 2.0mL: 25mL. 6. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that: the concentration of the phosphate buffer solution is 20 mmol, and the pH is 7.0; the polypeptide, phosphate The amount ratio of the buffer solution to the aldehyde-bonded silica gel is 80.0mg: 20mL: 2.0g. 7. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that: the concentration of the TFA solution is 80v%, and the amount of polypeptide-immobilized silica gel and TFA solution The ratio is 2.0g: 15mL. 8. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that: the functional monomer is trifluoromethacrylic acid, methacrylic acid or acrylamide ; The crosslinking agent is N'N-vinylbisacrylamide or N'N-methylenebisacrylamide; the initiator is ammonium persulfate; the concentration of phosphate buffer solution is 20mmol, and the pH is 7.0; - When ethylenebisacrylamide is used as the crosslinking agent, the dosage ratio of functional monomer, crosslinking agent, initiator and phosphate buffer solution is 126mg: 219mg: 10.3mg: 1.5mL, expressed as N'N-methylene When bisacrylamide is used as the crosslinking agent, the dosage ratio of functional monomer, crosslinking agent, initiator and phosphate buffer solution is 126mg: 30mg: 6.0mg: 1.5mL. 9. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that: the dosage ratio of the GGAKacR imprinted silica gel, acetone and HF solution is 0.5g: 10mL: 1.5mL. 10. The method for preparing molecularly imprinted microspheres for separating histone H4-K16 acetylation-marked polypeptides according to claim 2, characterized in that: the volume ratio of acetonitrile, acetic acid and distilled water in the acetonitrile-acetic acid-water mixed solution is 5:5:90. The volume ratio of acetonitrile, acetic acid and distilled water in the acetonitrile-acetic acid-water mixed solution is 5:5:90.