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Method for identifying glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrometry

A two-dimensional gel electrophoresis, glycoprotein technology, applied in the field of glycoproteomics, to achieve the effect of improving sensitivity and specificity

Inactive Publication Date: 2020-04-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention is a method for separating and identifying the sugar chain structure of a specific target glycoprotein biomarker, which has not been found in the same industry after searching.

Method used

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  • Method for identifying glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrometry
  • Method for identifying glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrometry
  • Method for identifying glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrometry

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Embodiment

[0031] Such as figure 1 As shown, this embodiment consists of the following steps:

[0032] S1. Specific enrichment of glycoproteins in saliva samples and separation by two-dimensional gel electrophoresis, and gel spots of target glycoprotein isomers are selected.

[0033]The saliva of healthy people was collected, refrigerated and centrifuged at 4°C, centrifugal force 2600g, 20min, and the supernatant was taken as saliva whole protein. Concanavalin A (Con A) affinity chromatography was used for glycoprotein enrichment. The agarose-bound lectin Con A was loaded into the column, and the binding buffer (20mM Tris-HCl, 150mM NaCl, 1mM CaCl 2 , 1mMMnCl 2 , 1 mM MgCl 2 , pH 7.4) for enrichment. Non-specifically bound proteins are then removed by washing the column with binding buffer. Finally, the elution buffer (0.2M α-methyl mannoside and 0.2M α-methyl glucoside added to the binding buffer) was used to release the enriched glycoproteins. The resulting glycoprotein was conc...

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Abstract

The invention discloses a method for identifying a glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrometry. The method comprises the following steps:S1, performing specific enrichment on glycoprotein in a biological sample, performing separation through two-dimensional gel electrophoresis, and selecting target glycoprotein isomer gel spots according to the molecular weight and isoelectric point of target glycoprotein; S2, confirming the selected target glycoprotein isomer gel spots by using a two-dimensional gel electrophoresis western blot and a mass spectrum; and S3, performing intra-gel enzymolysis and mass spectrometry on the target glycoprotein isomer gel spots confirmed in the step S2, and identifying the peptide fragment sequence and the sugar chain structure. The method is suitable for separation and structural identification of isomers and modifiers of various protein post-translational modified (glycosylated, ubiquitinated, acetylated and the like) biomarkers, and can significantly improve the sensitivity and specificity of the post-translational modified protein as a diagnostic marker.

Description

technical field [0001] The invention relates to the field of glycoproteomics, in particular to a method for identifying the sugar chain structure of a glycoprotein by combining two-dimensional gel electrophoresis and mass spectrometry; more specifically, it relates to a method for separating and identifying target glycoprotein isomers and identifying their sugar chain structure new method. Background technique [0002] Glycosylation is one of the most important post-translational modifications of proteins, which is involved in many key cellular processes, including cell recognition and adhesion, immune cell trafficking and carcinogenesis, etc. Glycosylation modification mainly occurs in the endoplasmic reticulum and Golgi apparatus. The main process is to transfer sugar chains to proteins under the action of glycosyltransferases, and form glycosidic bonds with amino acid residues on proteins. cleavage, modification and fucosylation or sialylation, etc., to complete the asse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/08G01N30/72
CPCG01N30/08G01N30/72
Inventor 肖华刘莎张岩
Owner SHANGHAI JIAO TONG UNIV
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