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Method of measuring glycosyl in protein

A protein and protease technology, applied in the field of biological protein detection, can solve the problems of difficult to preserve the sugar chain structure, incomplete sugar cleavage, poor reproducibility, etc., and achieve the effect of complete sugar chain structure and good repeatability

Active Publication Date: 2013-06-05
北京康弘生物医药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve a series of technical problems such as incomplete sugar cutting, difficulty in preserving the complete sugar chain structure, and poor reproducibility in the method for detecting sugar chains in proteins in the prior art, the present invention provides the following technical solutions:

Method used

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  • Method of measuring glycosyl in protein
  • Method of measuring glycosyl in protein
  • Method of measuring glycosyl in protein

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Experimental program
Comparison scheme
Effect test

Embodiment 3

[0127] 1. Experimental steps

[0128] 1) Liquid exchange process

[0129] Take 1mg of protein (sequence 1) solution, put it into an ultrafiltration centrifuge tube, supplement 400μl with 50mmol / L ammonium bicarbonate buffer (pH=8.3), centrifuge at 10000r / min for 10min, then add 350μl 50mmol / L ammonium bicarbonate buffer (pH=8.3), centrifuge at 10000 r / min for 10 min, and repeat the operation more than 3 times. At this time, the sample volume is about 40 μl, and the concentration is about 25 mg / ml.

[0130] 2) Sample digestion and sugar cutting

[0131] Take 10 μl of the protein (sequence 1) solution after liquid exchange treatment into a clean 1.5ml EP tube, and do two parallel experiments. Add an equal volume of 8M guanidine hydrochloride solution to the samples, mix well, and denature the protein in a 65°C water bath for 30 minutes; take the denatured sample, add 1 μl of 1M DTT solution to make the final concentration of DTT 50 mM, and reduce the protein in a 40°C water ba...

Embodiment 4

[0145] The detection of embodiment tetrasialic acid

[0146] Sialic acid is located at the outermost side of the sugar chain, and it is easy to fall off during the analysis process. In order to better reflect the pretreatment process and detection method of the present invention, the structure of the sugar chain is not changed, especially according to Example 1 (sample 1- 3) and Example 3 (sample 4-6), the sugar chains obtained by enzymatic cleavage and then sugar cleavage were used for detection of sialic acid.

[0147] The inventors determined the content of sialic acid in protein according to the resorcinol method recorded in the 2010 edition of "Chinese Pharmacopoeia", the third appendix VIC sialic acid determination method.

[0148] Calculate the content of sugar chains according to the HPLC results obtained by the method for detecting sugar groups in proteins according to the present invention, and the mass spectrometer detection results know the number of sialic acid in...

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Abstract

The invention relates to a method of measuring glycosyl in protein. The method specifically comprises that after being processed in a liquid-changing mode, the protein is cut by reducing ferment; a carbohydrate chain is directly cut by added carbohydrate-cutting enzyme; after the obtained carbohydrate chain is processed in a purifying mode, fluorescence derivation is conducted, and excessive markers are eliminated; and then the content of obtained carbohydrate chain is measured. The method of measuring the glycosyl in the protein can well guarantee that the carbohydrate is fully cut, and the carbohydrate chain is complete in structure and good in repeatability, and furthermore is simple and easy to operate.

Description

technical field [0001] The invention relates to the field of biological protein detection, in particular to a method for measuring sugar groups in proteins. Background technique [0002] Glycosylation refers to the process of attaching carbohydrates to proteins or lipids under the control of enzymes. It is an important post-translational modification of proteins. About half of the proteins are glycosylated. Glycoproteins The glycosyl can affect biological activity, mediate receptor recognition, increase solubility, regulate and stabilize protein conformation. Therefore, the special glycosyl structure is related to the safety and effectiveness of many protein drugs. It is of great significance to the biological activity, stability, immunogenicity, molecular transport and recognition of protein molecules. At the same time, the degree of protein glycosylation and Changes in the structure of sugar chains are often the hallmarks of tumors and other diseases. Therefore, in the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06
CPCG01N2333/924G01N2030/8831G01N33/68G01N30/00G01N33/50G01N2400/00
Inventor 邬智刚乔怀耀柯潇
Owner 北京康弘生物医药有限公司
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