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Nano gold doped integral material for enriching glycoprotein and applications thereof

A monolithic material and nano-gold technology, applied in the preparation method of peptides, other chemical processes, organic chemistry, etc., can solve the problems affecting the selectivity of enrichment, reduce the enrichment capacity, small specific surface area, etc., and achieve easy online enrichment collection, high enrichment selectivity, and the effect of increasing the specific surface area

Active Publication Date: 2014-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the specific surface area of ​​the overall material based on the silica gel matrix is ​​relatively large, but due to the effect of silanol, it has a strong non-specific adsorption on the protein, which will affect the selectivity of enrichment, and the pH range that the silica gel matrix material can tolerate Very narrow; and the main problem with the use of polymer matrix is ​​that its specific surface area is relatively small, thereby reducing the enrichment capacity

Method used

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  • Nano gold doped integral material for enriching glycoprotein and applications thereof
  • Nano gold doped integral material for enriching glycoprotein and applications thereof
  • Nano gold doped integral material for enriching glycoprotein and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Preparation of nano-gold doped monoliths modified by phenylboronic acid

[0024] Such as figure 1 As shown, it is prepared according to the following process:

[0025] 1) Preparation of monolithic material matrix containing epoxy groups: prepare the prepolymerization solution as follows: 240 mg glycidyl methacrylate, 160 mg polyethylene glycol diacrylate, 50 mg n-propanol, 550 mg cyclohexanol, and 4 mg Azodiisobutyronitrile. After the pre-polymerization solution was ultrasonicated for 10 minutes, the dissolved oxygen in the solution was removed by nitrogen gas; it was introduced into a capillary modified with double bonds, and the two ends of the capillary were sealed with silicone rubber, and the polymerization was initiated at 50°C. After 12 hours of polymerization time, respectively Flush out unreacted monomer and porogen with pure methanol and pure water;

[0026] 2) Nano-gold modification on the substrate surface: 1M cysteamine solution (dissolved in 1M NaOH ...

Embodiment 2

[0031] In order to investigate the enrichment capacity of phenylboronic acid-modified nano-gold doped monolithic column to glycoprotein, the material was determined by chromatographic frontier analysis. The specific measurement steps are as follows: 100mM phosphate buffer solution is used as loading buffer, 1M ammonium bicarbonate solution that is not retained on the column (Example 1) is used to measure the dead time, and then 0.2M HRP is continuously passed into the affinity column, Measure its peak time, thereby calculate the adsorption capacity of this column to glycoprotein HRP to be 2.08mg / g.

Embodiment 3

[0033] In order to investigate the selectivity and non-specific adsorption of phenylboronic acid-modified nano-gold doped monolithic column for glycoprotein enrichment, further interference experiments were carried out.

[0034] 1. Protein mixed solution preparation

[0035] Glycoprotein (horseradish peroxidase, HRP) and non-glycoprotein (bovine serum albumin, BSA) were mixed at a mass ratio of 1:1000 and dissolved in 50 mM ammonium bicarbonate buffer solution (pH 10.0) to prepare A protein mixed solution with a concentration of 10.01 mg / mL was obtained.

[0036] 2. Selective enrichment of glycoproteins

[0037] The phenylboronic acid-modified nano-gold doped monolithic column (150 μm inner diameter, 10 cm long) prepared in Example 1 was equilibrated with 50 mM ammonium bicarbonate buffer solution (pH 10.0) for 20 min. at 0.5μL·min -1 The above-mentioned protein mixture solution was passed into the column at a flow rate of 20 min, and the column was washed with 50 mM ammoni...

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Abstract

The invention relates to a preparation method of a nano gold doped integral material for enriching glycoprotein. The preparation method comprises the following steps: taking glycidyl methacrylate (GMA) and polyethylene glycol diacrylate (PEGDA) as the monomers to synthesize a hydrophilic polymer integral material substrate through an in-situ polymerization method; utilizing the epoxy groups in the substrate surface to enrich the substrate surface with mercapto groups through a chemical derivatization method, then modifying the substrate surface with nano gold particles; finally co-modifying 4-mercaptophenylboronic acid and mercaptoethylamine on the nano gold surfaces on the substrate, and enriching glycoprotein on the basis of phenylboronic acid affinity chromatography. The hydrophilicity of the monomer polyethylene glycol diacrylate (PEGDA) is utilized so as to reduce the non-specific adsorption of substrate on proteins, and thus the enrichment selectivity is improved. At the same time, the high specific surface area of nano particles is utilized at the same time, thus the enrichment capacity is increased, and finally the high-efficient and high-selective enrichment of glycoprotein is achieved.

Description

technical field [0001] The invention relates to the enrichment of glycoproteins by a functionalized monolithic material, in particular to the preparation of a nano-gold doped monolithic material and its use for high-efficiency and high-selectivity enrichment of glycoproteins. Background technique [0002] Protein glycosylation is an important post-translational modification. According to reports, at least 50% of proteins are glycosylated, which has an important impact on protein structure and function. However, due to the low abundance of glycoproteins and the small proportion of glycopeptides in the total peptides, they are often covered by high-abundance non-glycopeptides when detected by mass spectrometry. Analyzing glycoproteins is nearly impossible. The commonly used separation and enrichment methods for glycoproteins reported in the literature mainly include lectin affinity method, boric acid method, hydrazide method, and hydrophilic interaction method. The boric aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/26B01J20/30C07K1/22
Inventor 张丽华吴慈梁玉杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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