Method for separating and identifying glycoprotein N-sugar chain structure

A technology for identifying sugars and glycosylated proteins, which is applied in material separation, analysis materials, measurement devices, etc., can solve the problems of low kurtosis N-sugar chain information loss, inability to obtain N-sugar chain information, etc. Inhibitory effect, high sensitivity, rich sugar chain information effect

Inactive Publication Date: 2014-11-05
苏州中科纳泰生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when all N-glycans in some samples were detected by mass spectrometry, it was found that they contained very rich high-mannose N-glycans and less complex and heterozygous N-glycans. On the spectrum, the peak The high mannose N-glycan

Method used

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  • Method for separating and identifying glycoprotein N-sugar chain structure
  • Method for separating and identifying glycoprotein N-sugar chain structure
  • Method for separating and identifying glycoprotein N-sugar chain structure

Examples

Experimental program
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Embodiment 1

[0026] Example 1 Analysis of N-sugar chain structure of chicken ovalbumin

[0027] 1. Using ConA to separate and enrich high mannose N-glycans and non-high mannose N-glycans

[0028] (1) Separation and enrichment of non-high mannose N-glycan chains:

[0029] Take 100 μg of standard protein (chicken ovalbumin) and put it into an ultrafiltration tube, directly add 300 μL of 8M urea, mix well, and centrifuge at 14000 g for 15 min. Add 200 μL of 8M urea solution to the ultrafiltration tube, and centrifuge at 14000 g for 15 min. Discard the effluent in the collection tube, add 150 μL of 10 mM DTT and mix well, incubate at 56 ° C for 45 min, centrifuge at 14000 g for 10 min, add 150 μL of 20 mM IAM and mix well, incubate in the dark for 20 min, and centrifuge at 14000 g for 10 min. Discard the effluent from the collection tube. Add 150 μL coupling buffer (0.1mol / L Tris-HCl, 150mmol / L NaCl, 1mmol / L CaCl 2 , 1mmol / L MgCl 2 , 1mmol / L MnCl 2 , pH7.4) into an ultrafiltration tube, ce...

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Abstract

The invention discloses a method for separating and identifying glycoprotein N-sugar chain structure, and belongs to the technical field of sugar chain detection. According to the method, concanavalin (ConA) is used to separate and enrich high-mannose type and non-high-mannose type N-sugar chains; matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS) is used to detect; a signal-to-noise ratio of 6 is used as a threshold value to carry out data screening; and a Glycoworkbench software is used to analyze. Structure shows that 23 N-sugar chains are identified from a reference group; 29 N-sugar chains are identified from two parts of the high-mannose type and non-high-mannose type; and the identified N-sugar chains are six more than that before ConA enrichment. The method has higher sensitivity and more abundant sugar chain information and has obvious advantage for identification of low kurtosis sugar chains.

Description

technical field [0001] The invention relates to a method for separating and identifying the N-sugar chain structure of glycoproteins, especially a method for separating, enriching and identifying high-mannose and non-high-mannose N-sugar chains with concanavalin lectin, which belongs to the sugar chain detection technology field. Background technique [0002] As the most common post-protein modification, glycosylation is closely related to the structure and function of proteins, affecting the folding of proteins, and then affecting their own biological functions. Abnormal glycosylation is associated with many diseases, especially closely related to the occurrence and development of various cancers. N-glycans are an important biomarker in the development of cancer. Recent studies have shown that increased fucosylation and decreased sialylation of N-glycans were detected in the serum of patients with advanced breast cancer; Increased sialylated Lewis structures were detected...

Claims

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Application Information

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IPC IPC(8): G01N30/72G01N30/08
Inventor 关锋杨刚龙谭增琦李想郭佳庞星辰
Owner 苏州中科纳泰生物科技有限公司
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