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Preparation method of glycoprotein microreactor for boron affinity surface imprinting of mesoporous molecular sieve

A technology of mesoporous molecular sieve and microreactor, applied in the field of glycoproteomics analysis

Active Publication Date: 2021-08-27
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there is no literature report on a glycoprotein microreactor that specifically recognizes glycopeptides based on molecular imprinting technology, especially providing a glycoprotein microreactor that integrates protein extraction and enzymatic digestion and specifically enriches N-glycopeptide functions. The preparation method of the reactor is specifically to use SBA-15 (SBA series, Santa Barbara Amorphous) mesoporous molecular sieve as the carrier, 2,3-difluoro-4-formylphenylboronic acid as the monomer, and N-acetylneuraminic acid Preparation of molecular sieve-based surface-directed imprinted polymer (SBA-15@MIP) for fragment templates

Method used

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  • Preparation method of glycoprotein microreactor for boron affinity surface imprinting of mesoporous molecular sieve
  • Preparation method of glycoprotein microreactor for boron affinity surface imprinting of mesoporous molecular sieve
  • Preparation method of glycoprotein microreactor for boron affinity surface imprinting of mesoporous molecular sieve

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Experimental program
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Effect test

Embodiment 1

[0034] The preparation of mesoporous molecular sieve boron affinity surface imprinted polymer (SBA-15@MIP), this example includes the following five steps:

[0035] (1) Pretreatment of SBA-15 molecular sieve

[0036] SBA-15 (Shanghai Aladdin Reagent Co., Ltd.) was activated in HCl solution to obtain more hydroxyl groups. The specific operation is as follows, disperse 200 mg of SBA-15 into 8 mL of 6 M HCl, and stir at room temperature for 10 hours. Then, the supernatant was discarded by centrifugation, and the SBA-15 was washed five times with deionized water until the pH of the supernatant was neutral. Secondly, the acidified SBA-15 was vacuum-dried at room temperature, and then vacuum-dried at 110° C. for 6 hours.

[0037] (2) Amino functionalization of SBA-15 mesoporous molecular sieve materials

[0038] Amino-functionalized SBA-15 was prepared using APTES (3-aminopropyltriethoxysilane). First, 200 mg of SBA-15 was uniformly dispersed in a 100 mL glass flask containing 5...

Embodiment 2

[0047] The extraction performance of SBA-15@MIP on template molecules in actual samples, such as figure 2 .

[0048] To further evaluate the extraction performance of the obtained mesoporous molecular sieve boron affinity surface-imprinted polymer (SBA-15@MIP) for template molecules in real samples, SBA-15@MIP was used to detect N-acetyl Extraction recovery of neuraminic acid. The present invention investigates the extraction performance of normal human serum samples added with different concentrations of N-acetylneuraminic acid. The specific operation steps are as follows:

[0049] (1) Synthesize SBA-15@MIP with the above method (Example 1).

[0050] (2) In this experiment, SBA-15@MIP was coated on the surface of glassy carbon electrode (GCE) by drop coating method. The specific steps are as follows: First, 1.0, 0.3 and 0.05 μM of GCE alumina slurry were applied on the polishing cloth respectively. The glassy carbon electrode was polished, and then ultrasonically cleaned...

Embodiment 3

[0057] The effect of the amount of SBA-15@MIP added on protein adsorption in serum samples, such as image 3 . Specific steps are as follows:

[0058] (1) Weigh 1.5-12 mg of SBA-15@MIP and place it in a centrifuge tube. Afterwards, 2 μL of serum to be treated was added, and 48 μL of Tris-HCl solution (pH 8.0, 50 mM) was added to wet the material, and the supernatant was obtained by centrifugation after shaking (room temperature).

[0059] (2) Quantitative analysis was carried out according to the Bradford method. Mix the dye Coomassie blue G-250 and the sample to be tested at a volume ratio of 10:1, and then incubate at room temperature for 2 minutes to facilitate thorough mixing, and then measure the absorbance at 595 nm with a UV-3310 spectrophotometer, record . Finally, the percent protein adsorbed was calculated from the standard curve of the protein.

[0060] According to the obtained data, draw the corresponding image 3 . When 2 μL of human serum samples were add...

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Abstract

The invention relates to a preparation method of a glycoprotein microreactor for boron affinity surface imprinting of a mesoporous molecular sieve. According to the method, an SBA-15 mesoporous molecular sieve is used as a carrier, 2,3-difluoro-4-formylphenylboronic acid is used as a monomer, and N-acetylneuraminic acid is used as a fragment template to prepare the molecular sieve-based surface oriented imprinting polymer (SBA-15-coated MIP). According to the micro-reactor, protein in a sample is extracted by utilizing a size exclusion effect, the protein is subjected to rapid enzymolysis based on a nano confinement effect, and a peptide fragment containing a sugar chain is selectively enriched by utilizing a specific recognition effect of molecular imprinting. According to the method, the used materials are low in price and easy to obtain, preparation is easy, operation is easy and convenient, the three sample pretreatment processes of protein extraction, rapid protein enzymolysis and glycopeptide enrichment can be integrated, and the pretreatment time of the glycoproteomics sample can be effectively shortened.

Description

technical field [0001] The invention belongs to the field of glycoproteomics analysis, and in particular relates to a method for preparing a glycoprotein microreactor imprinted on the boron-affinity surface of a mesoporous molecular sieve. The invention can complete the three sample pretreatment processes of fast protein extraction, enzymatic hydrolysis and glycopeptide enrichment, and has less sample loss and higher analysis sensitivity compared with the currently commonly used N-glycoproteomics analysis method , and can significantly shorten the time required to obtain glycopeptides from serum samples, and has the characteristics of rapidity, simplicity, and low sample loss. Background technique [0002] Glycosylation is one of the most common protein translational modifications (Protein translational modifications, PTMs) and is required for survival in mammals. It is estimated that approximately 50% of human proteins are glycosylated. Abnormal glycosylation is closely r...

Claims

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Application Information

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IPC IPC(8): B01J19/00B01J20/26B01J20/30C12P21/06C07K1/22C07K1/14
CPCB01J19/0093B01J20/268B01J20/3057B01J20/3071B01J20/3085C12P21/06C07K1/22C07K1/14B01J2219/00918
Inventor 刘照胜袁芳芳黄艳萍
Owner TIANJIN MEDICAL UNIV
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