Method for separation, analysis and identification of reductively released glycoprotein n-glycan and its derivatives

A technology for glycoproteins and derivatives, applied in the field of glycobiology, can solve problems such as easy decomposition, unstable N-glycan chains, unfavorable analysis, etc., and achieve the effects of less side reactions, improved detection sensitivity, and strong versatility.

Active Publication Date: 2020-04-10
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In order to solve the lack of a general chemical method for releasing N-sugar chains containing core α-1,3-fucose glycosylated N-sugar chains in the prior art, and the N-sugar obtained by the existing method Sugar chains are unstable and easy to decompose, which is not conducive to the defects of subsequent analysis. The present invention provides a method for reductively releasing N-sugar chains of glycoproteins

Method used

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  • Method for separation, analysis and identification of reductively released glycoprotein n-glycan and its derivatives
  • Method for separation, analysis and identification of reductively released glycoprotein n-glycan and its derivatives
  • Method for separation, analysis and identification of reductively released glycoprotein n-glycan and its derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1: Release and purification of egg albumin N-sugar chains by reducing chemical method

[0124] Chemical release:

[0125] Weigh 10 mg of egg albumin, which is a neutral N-sugar chain, dissolve it in concentrated ammonia water (1 mL, concentration 26% to 28%); add NaOH solid to the reaction system until the concentration of NaOH in the reaction system is 0.3mol / L, add NaBH to the reaction system 3 CN solid to NaBH in the reaction system 3 The concentration of CN was 1mol / L; the obtained reaction mixture was reacted at 40°C for 16 hours. After the reaction, the reaction system was concentrated under reduced pressure with a rotary evaporator, and after redissolving with 1 mL of double distilled water, the pH was adjusted to neutral (pH 7 or so), again use a rotary evaporator to concentrate under reduced pressure to remove the solvent, and obtain the crude N-sugar chain of egg albumin. For neutral N-sugar chains, adding sodium hydroxide in the reaction system c...

Embodiment 2

[0140] Example 2: Release and purification of buckwheat pollen total protein N-glycans by reducing chemical method

[0141] Chemical release:

[0142] Weigh 10 mg of buckwheat pollen total protein, which is a neutral N-sugar chain, dissolve it in concentrated ammonia water (1 mL, mass concentration 26% to 28%); add NaOH solid to the reaction system The concentration of NaOH in the medium is 0.3mol / L, add NaBH to the reaction system 3 CN solid to NaBH in the reaction system 3 The concentration of CN is 1mol / L. The obtained reaction mixture was reacted at 40°C for 16 hours. After the reaction was completed, the reaction system was concentrated under reduced pressure with a rotary evaporator, and 1 mL of double-distilled water was added to dissolve it again. Concentrate under reduced pressure with a rotary evaporator to remove the solvent to obtain the crude N-glycan chain of buckwheat pollen total protein.

[0143] The method for purifying the crude N-sugar chain of buckwh...

Embodiment 3

[0150] Example 3: Release and purification of fetal bovine serum total protein N-glycans by reducing chemical method

[0151] Chemical release:

[0152] Weigh 10 mg of fetal bovine serum total protein, the sugar chain of fetal bovine serum total protein is acidic N-sugar chain, dissolve in concentrated ammonia water (1mL, concentration 26%-28%); add NaBH to the reaction system 3 CN solid to NaBH in the reaction system 3 The concentration of CN is 1mol / L. The obtained reaction mixture was reacted at 40°C for 16 hours. After the reaction was completed, the reaction system was concentrated under reduced pressure with a rotary evaporator, and 1 mL of double-distilled water was added to dissolve it again. Concentrate under reduced pressure with a rotary evaporator to remove the solvent to obtain the crude product of N-sugar chains of fetal bovine serum total protein.

[0153] For acidic N-sugar chains, no sodium hydroxide is added to adjust the alkalinity to prevent deacetylat...

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Abstract

The invention belongs to the field of glycobiology technology, and specifically relates to a method for the preparation, separation, analysis and identification of N-sugar chains of reductively released glycoproteins and their derivatives. The method for reductively releasing glycoprotein N-sugar chains of the present invention is to first dissolve the glycoprotein in concentrated ammonia water to hydrolyze the sugar chains, and then pass it through NaBH 3 The reduction of CN yields an active amino group (NH 2 ) of sugar amines, this solution is highly versatile and is suitable for neutral N-glycans, acidic N-glycans and neutral N-glycans containing core α-1,3-fucosylation, releasing N-sugar chains are highly stable and will not degrade, allowing direct preliminary analysis. In addition, the present invention provides a method for labeling the obtained N-sugar chain with Fmoc, and also provides a method for separating, analyzing and identifying the N-sugar chain and the Fmoc-labeled N-sugar chain.

Description

technical field [0001] The invention belongs to the technical field of glycobiology, and in particular relates to a method for reductively releasing glycoprotein N-sugar chains and derivatives thereof for separation, analysis and identification. Background technique [0002] Glycosylation is one of the most important post-translational modifications of proteins, and plays a very important role in protein translation regulation and protein degradation. More than 50% of proteins exist in the form of glycoproteins, and more and more studies have shown that abnormal glycosylation can lead to human diseases. In the course of clinical treatment, the antibody drugs used are all glycosylated. [0003] Glycosylation can be roughly divided into four types according to the way proteins are linked to sugar chains: N-linked glycosylation, O-linked glycosylation, C-mannose glycosylation, and glycosylphosphatidylinositol ( glycophosphatidlyinositol, GPI) anchors the connection. O-linked...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N27/62
CPCG01N27/62G01N30/02G01N30/06
Inventor 王仲孚王承健强珊黄琳娟
Owner NORTHWEST UNIV
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