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Preparation and application of multi-component block hydrophilic copolymer-silica gel hybrid chromatography packing

A block copolymer and silica gel hybrid technology, applied in the field of material chemistry, can solve the problems of low affinity between lectin and sugar chain, low enrichment efficiency, general selectivity, etc., achieve obvious selective enrichment effect and improve identification Effects of Quantity, Increased Efficiency and Selectivity

Inactive Publication Date: 2018-03-02
BEIJING PROTEOME RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low affinity between lectins and sugar chains, the selectivity is average, so the enrichment efficiency is low

Method used

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  • Preparation and application of multi-component block hydrophilic copolymer-silica gel hybrid chromatography packing
  • Preparation and application of multi-component block hydrophilic copolymer-silica gel hybrid chromatography packing
  • Preparation and application of multi-component block hydrophilic copolymer-silica gel hybrid chromatography packing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Such as figure 1 As shown, the process for preparing multi-component block hydrophilic copolymer-silica gel hybrid packing:

[0040] 1) Synthesis of 2-3-glucosaminopropyl methacrylate (GMAG) or 3-mannosylaminopropyl methacrylate (GMAM) monomer: Into 674.53 μl glycidyl methacrylate (GMA) Add 0.2M sulfuric acid, place it in a water bath at 50°C and heat it for oxidation reaction for 4 hours, and then obtain the oxidized GMA that converts epoxy groups into aldehyde groups;

[0041] Measure another 20 ml of methanol, gradually add 1.1 g of glucosamine or mannose, and stir until dissolved. Add the obtained oxidized GMA dropwise to the continuously stirring methanol solution of glucosamine or mannose amino, stir at room temperature to carry out Schiff base reaction for 4 hours to obtain a yellow transparent solution, and blow dry the prepared solution with nitrogen until it becomes a paste , to obtain GMAG or GMAM monomer, sealed and filled with nitrogen, then stored at 4°C...

Embodiment 2

[0053] Utilize the chromatographic filler obtained in Example 1 to carry out the enrichment of glycopeptides:

[0054] 1) Dissolve 10 mg of the chromatographic filler prepared in Example 1 with acetonitrile, fill it into a 200 µl pipette tip, and precipitate naturally to prepare a solid phase extraction column;

[0055] 2) Weigh an appropriate amount of asialo-bovine fetuin / bovine serum albumin and dissolve it in 50 mM ammonium bicarbonate solution to a final concentration of 1 μg / μl. Add mercaptoethanol at a final concentration of 10 mM, reduce in a water bath at 56°C for 1 hour, then add IAA and place in the dark for 1 hour to denature the protein. Take the denatured protein and add trypsin at a mass ratio of 1:50 (trypsin:protein), place it in a 37°C water bath and incubate for 12 hours, then add 0.1% TFA to inactivate the trypsin to obtain an enzymatic hydrolysis product.

[0056]The asialo bovine fetuin and bovine serum albumin peptide products obtained after the enzymol...

Embodiment 3

[0062] The enrichment of glycopeptides in mouse liver whole protein was carried out by using the multi-component copolymer-silica gel hybrid chromatographic filler obtained in Example 1:

[0063] 1) Dissolve 10 mg of the chromatographic filler prepared in Example 1 with acetonitrile, fill it into a 200 μl pipette tip, and precipitate naturally to prepare a solid phase extraction column;

[0064] 2) Take 50 μg of mouse liver whole protein and dissolve it in 50mM ammonium bicarbonate solution with a final concentration of 2 μg / μl. After thermal denaturation in a boiling water bath for 10 minutes, cool to room temperature, add an appropriate amount of denatured protein to PNGase F enzyme (the mass ratio of enzyme and protein is 1:10), and incubate in a 37°C water bath for 16 hours. The enzymatic hydrolyzate was lyophilized and dissolved in acetonitrile / water / formic acid (80:20:0.1, v / v) with a final concentration of 2 μg / μl. The enrichment process is as follows:

[0065] (1) Ma...

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Abstract

The invention discloses the preparation and application of a multi-component block hydrophilic copolymer-silica gel hybrid chromatographic packing. Polymerize SBMA, GMAG, GMAM and other hydrophilic monomers to generate multi-component block hydrophilic copolymer-silica gel hybrid fillers. The surface of the hybrid filler is highly rough, so it has a high specific surface area. The solid-phase extraction column and high-performance liquid chromatography column packed with this multi-component block hydrophilic copolymer-silica gel hybrid filler successfully achieved the enrichment of glycopeptides and sugar chains in standard glycoproteins and complex samples of mouse liver proteins Collection, separation, selective enrichment, and separation effect are obvious. A total of 1244 glycopeptides were enriched and identified in mouse liver proteins, corresponding to 577 glycoproteins, fully proving its role in the glycosylation analysis of complex protein samples. Great application value.

Description

technical field [0001] The invention relates to the field of material chemistry, in particular to the preparation and application of a multi-component block hydrophilic copolymer-silica gel hybrid chromatographic filler. Background technique [0002] Protein glycosylation is one of the most common and complex post-translational modifications, which regulates many important physiological functions in organisms, such as protein transport, localization and interaction, as well as recognition and information transmission between cells. Current research has shown that the abnormality of protein glycosylation expression level and glycosylation degree is closely related to the occurrence and development of many tumors. At present, through the research on the function of some glycoproteins and the identification of glycosylation sites, many glycoproteins or sugar chains have become disease diagnostic markers and targets for new drug development. 50% of the tumor diagnostic markers ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/283B01J20/286B01J20/30C08F293/00C07K1/14C07K1/16C07H3/06C07H1/06
Inventor 钱小红秦伟捷潘一廷张万军
Owner BEIJING PROTEOME RES CENT
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