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Glycopeptide or glycoprotein enrichment material, preparation thereof and application of material

A glycoprotein enrichment technology, applied in the application field of glycoproteomics, can solve the problems of inapplicable O-type glycosylation site identification, lack of glycosidase, etc., and achieve mild reaction conditions and good particle dispersion , a wide range of effects

Inactive Publication Date: 2019-03-19
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

However, due to the lack of suitable glycosidases, the hydrazide method is not suitable for the identification of O-glycosylation sites

Method used

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  • Glycopeptide or glycoprotein enrichment material, preparation thereof and application of material
  • Glycopeptide or glycoprotein enrichment material, preparation thereof and application of material
  • Glycopeptide or glycoprotein enrichment material, preparation thereof and application of material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Preparation of mercaptopolymer spheres: In a 5mL centrifuge tube, add 2mL of phosphate buffer solution PB (50mM, pH8), add 30μg of polystyrene particles with a particle size of 10μm and aldehyde groups on the surface, ultrasonically disperse evenly, Add 200mg of CD (2,2'-dithiodiethylamine), 10mg of sodium cyanoborohydride, react with a constant temperature oscillator at 30°C for 24h, and stop the reaction. Centrifuge at a speed of 3000 rad / min in a high-speed centrifuge, remove the supernatant, wash with water and Tris-HCl buffer solution with pH 8 in turn for 4 times, dry in a vacuum oven at 50°C for 24 hours, and redisperse in phosphate buffer solution Add 0.1mg of tris(2-carboxyethyl)phosphine (TCEP) to PB (50mM, pH 8), shake and react at 40°C for 1h, centrifuge at a speed of 3000rad / min in a high-speed centrifuge, remove the supernatant, and then use Wash 3 times with water and dry in a vacuum oven to constant weight. Polymer particles modified with mercapto group...

Embodiment 2

[0023] Preparation of mercaptosilica spheres: In a 150mL round bottom flask, add 50mL of toluene, add 2.5g of silica gel particles with a particle size of 5μm, and 1ml of 3-mercaptopropyltriethoxysilane, and disperse evenly by ultrasonication for 2 minutes. A condenser was connected to the flask, and mechanically stirred at a speed of 300 rad / min. The reaction device was placed in an oil bath, heated to reflux at 110°C for 15 hours, the reaction was stopped, and cooled to room temperature. Centrifuge at a speed of 3000 rad / min in a high-speed centrifuge, remove the supernatant, use toluene, acetone, methanol, and acetone to filter and wash successively, repeat the suction and filter washing for 3 times, and dry in a vacuum oven at 50°C for 16 hours to obtain Silica gel particles modified with mercapto groups.

Embodiment 3

[0025] Preparation of hydrazide microspheres: Disperse 25 mg of the mercapto microsphere particles obtained in Example 1 in a PB buffer solution of pH 7.8, and disperse evenly by ultrasonication, add 10 mg of PDPH (3-(2-pyridyldithio)propionyl hydrazide), and disperse by ultrasonication , shake and react at 30°C for 16 hours; then use a high-speed centrifuge to centrifuge at a speed of 3500rad / min for 5min, remove the supernatant, wash the microspheres with reaction solvent, water, and 50mM sodium acetate in sequence, repeat the washing 3 times, and dry in vacuum In the box to constant weight, the sugar chain-releasable glycosylated peptide / protein enrichment material is obtained. The preparation process is as figure 2 shown.

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Abstract

The invention belongs to the technical field of glycopeptide / glycoprotein enrichment materials and analysis, and relates to a sugar chain releasable glycosylated peptide fragment / protein enrichment material based on a hydrazide strategy, a preparation method of the material and an application of the material. The surface of a matrix micro-sphere is bonded with a hydrazide compound containing disulfide bonds to form a functional material with a hydrazide group on the surface, wherein the functional material can be used for glycopeptide enrichment. The disulfide bonds are led to the surface of the material to realize sugar chain releasable enrichment of glycopeptide / glycoprotein, sugar chains are in covalent binding, and the material has the advantage of high enrichment efficiency and overcomes the shortcoming that the sugar chains are not easily released in a traditional hydrazide method and cannot be used for O-glycosylated peptide fragment / protein enrichment. The sugar chain releasable enrichment material is easy to prepare and good in enrichment selectivity, is widely applicable to N-glycosylated and O-glycosylated peptide fragment / protein enrichment and has a high practical value in the fields of glycoproteomics and the like.

Description

technical field [0001] The invention relates to a sugar chain-releasable glycosylated peptide / glycoprotein enrichment material based on a hydrazide strategy, a preparation method thereof, and an application thereof in glycoproteomics. Background technique [0002] Protein glycosylation is one of the most important and common post-translational modifications in organisms, and is closely related to biological processes and physiological functions such as cell adhesion, signal transduction, apoptosis, and immune response (Rudd, et al. Science 2001, 291, 2376-2378). As an important biomarker, glycoprotein has been widely used in disease diagnosis, early warning and drug efficacy evaluation (Overath, et al. Mol. Cell. Proteomics 2012, 11, 467-477). In view of the significance of glycosylation modification in life activities, glycosylation proteomics, as an important branch of proteomics, has also received more and more attention. However, the research object of glycosylation pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/22B01J20/26B01J20/28B01J20/30C07K1/14
CPCB01J20/103B01J20/22B01J20/265B01J20/28004B01J20/28019C07K1/145
Inventor 张丽华邵文亚梁玉杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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