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A kind of glycoprotein enrichment and purification method

A purification method and protein technology, which are applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., can solve the problems of large loss of enriched glycoprotein samples, inability to replace solvents, and complicated steps, so as to reduce the loss of samples. , Easy to operate, efficient enrichment effect

Active Publication Date: 2017-11-14
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

[0005] In order to overcome the shortcomings of the hydrazide chemical method for enriching glycoproteins, such as large loss of samples, cumbersome steps, and inability to replace solvents, the present invention provides a method for enriching and purifying glycoproteins, which can complete hydrazide enriching glycoproteins in situ on a centrifugal ultrafiltration device. This method has the advantages of small sample loss, high enrichment efficiency, easy operation, and the simultaneous enrichment of glycoproteins in soluble proteins and / or insoluble membrane proteins

Method used

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  • A kind of glycoprotein enrichment and purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Oxidation of Glycoproteins

[0023] The whole protein sample extracted from rat liver was dissolved in 10mM sodium periodate solution prepared by 4% SDS oxidation buffer (100mM sodium acetate, 150mM sodium chloride, pH5.5), and the sample solution was added to the ultrafiltration React for 1 h in the ultrafiltration tube of a centrifugal ultrafiltration device with a membrane pore size of 0.45um (purchased from Pall Corporation, Port Washington, USA);

[0024] 2. Removal of excess oxidants

[0025] After the oxidation reaction is over, add oxidation buffer solution to the ultrafiltration tube to prepare a 20 mM sulfite solution for 10 minutes to neutralize excess oxidant;

[0026] 3. Capture of Glycoproteins

[0027] Add agarose gel with hydrazide groups suspended in the oxidation buffer to the ultrafiltration tube, and shake at room temperature for 12 hours;

[0028] 4. Protein denaturation and reduction

[0029] The sample processed in step 3 was centrifuged at...

Embodiment 2

[0047] The whole protein sample extracted from rat liver in Example 1 was replaced with the whole protein sample extracted from human cervical cancer cells; the enrichment steps of the glycoprotein sample were the same as in Example 1.

[0048] Identification result

[0049] total protein number identified

Embodiment 3

[0051] The steps of the oxidation method of glycoproteins are as follows: the 10mM sodium periodate solution prepared with 1% Triton X-100 oxidation buffer solution (100mM sodium acetate, 150mM sodium chloride, pH5.5) is used to dissolve the glycoprotein extracted from rat liver. Whole protein sample; glycoprotein sample enrichment steps are the same as in Example 1.

[0052] Identification result

[0053] total protein number identified

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Abstract

The invention relates to a method for enriching and purifying glycoproteins. The method combines the hydrazide chemical method for highly selective enrichment of glycoproteins and the use of a centrifugal ultrafiltration device for high-efficiency purification. The hydrazide is completed in situ on the centrifugal ultrafiltration device. Enrichment glycoprotein process. This method carries out the whole glycoprotein enrichment process in situ, which can effectively reduce the sample loss. At the same time, the use of centrifugal ultrafiltration device makes it possible to apply solubilizer in the enrichment process, so as to achieve soluble protein and / or insoluble membrane protein Simultaneous enrichment of glycoproteins; the advantages of effective replacement of solvents by centrifugal ultrafiltration devices enable the introduction of various types of elution solutions during the enrichment of glycoproteins to complete the effective removal of non-glycoproteins, thereby improving the selectivity of glycoprotein enrichment.

Description

technical field [0001] The invention relates to the field of biological analysis, in particular to a method for enriching and purifying glycoproteins. Background technique [0002] Glycosylation is one of the most complex protein post-translational modifications, and more than 50% of proteins in mammals are predicted to be glycosylated modified proteins. Glycosylated proteins have rich biological functions including signal transduction, cell adhesion, and immune response; the abnormality of protein glycosylation is closely related to tumors, immunodeficiency, and degenerative diseases; , an important target of biomarkers of drug response, therefore, research and development of new technologies and methods for high-throughput analysis of glycoproteins in complex samples has become a research hotspot. However, due to the low content of glycopeptides, the easy suppression of mass spectrometry signals of glycopeptides, and the microscopic heterogeneity of glycoforms, the curren...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/34
Inventor 张丽华方菲赵群杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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