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Method for identifying simplified O-GalNAc glycosylation peptide fragment based on chemical reaction

A technology of chemical reaction and identification method, which is applied in the field of mass spectrometry acquisition in the field of proteomics research, can solve the problems of not reflecting the real state of cells, complex, unsuitable tissue and body fluid samples, etc., and achieves low price, short reaction time and low cost Effect

Inactive Publication Date: 2020-02-04
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

But this method requires complex genetic engineering steps, does not reflect the true state of the cells, and is not suitable for tissue and body fluid samples

Method used

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  • Method for identifying simplified O-GalNAc glycosylation peptide fragment based on chemical reaction
  • Method for identifying simplified O-GalNAc glycosylation peptide fragment based on chemical reaction
  • Method for identifying simplified O-GalNAc glycosylation peptide fragment based on chemical reaction

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Embodiment 1

[0024] Low-abundance peptide analysis method in micro-system for proteomics analysis of human serum:

[0025] (1) Dissolve human serum in a buffer solution containing 8M Urea and 50mM HEPES, add a final concentration of 20mM MDTT, react in a water bath at 37°C for 2h, then add IAA with a final concentration of 40mM, and react in the dark at 25°C for 40min;

[0026] (2) For the sample obtained after step (1), dilute the concentration of Urea to 1 M with an aqueous solution of 50 mM HEPES, then add Trypsin with a mass ratio of 1 / 20 to the protein, and enzymolyze it in a water bath at 37°C for 20 h, Obtain peptide solution;

[0027] (3) Adjust the pH value of the sample obtained after step (2) to about 2 with TFA, and select the corresponding C 18 The solid-phase extraction column removes small molecules in the solution, and the peptide eluate is freeze-dried;

[0028] (4) Redissolve the peptide obtained in step (3) in 50 mM sodium phosphate solution (pH=7.5), add PNGase F with...

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Abstract

The invention relates to a method for simplifying an O-GalNAc glycosylation structure based on chemical reaction. The sialic acid at the tail end of O-GalNAc glycosylation greatly increases the structural complexity of glycosylation, the characteristic that sialic acid residues at the tail end of the glycosylation peptide fragment are prone to hydrolysis under acidic and heating conditions is utilized, and a certain proportion of organic acid is adopted as an acidic matrix, so that rapid hydrolysis of the sialic acid residues is achieved, the removal efficiency of the sialic acid residues reaches 95% or above, and the structural complexity of glycosylation is remarkably reduced. Moreover, the mass spectrum fragmentation efficiency and the detection sensitivity of the simplified O-GalNAc glycopeptide are more efficient, and the effect of the O-GalNAc glycopeptide is remarkably superior to that of currently commercialized sialidase activity when the O-GalNAc glycopeptide is used for large-scale analysis of O-linked glycosylation.

Description

technical field [0001] The invention belongs to the field of mass spectrometry acquisition methods in the direction of proteomics research, and specifically relates to the combination of hydrophilic-affinity chromatographic enrichment technology and acid-assisted O-sugar chain sugar type simplification technology to realize efficient detection of mucin-type O-sugar in human serum samples Kylated peptides. Background technique [0002] As an important type of glycosylation modification, mucin-type O-glycosylation (Mucin-type, O-GalNAc) is involved in various life processes in vivo such as cell adhesion, cell signal transduction and immune response. Abnormal O-GalNAc glycosylation levels are associated with many important diseases, so the research on O-GalNAc is very important (document 1. Fukuda, M., Roles of mucin-type O-glycans in cell adhesion. Bba - Gen Subjects 2002, 1573, 394-405). However, compared with the relatively mature N-linked glycosylation and O-linked nitrog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N30/02
CPCG01N30/02G01N33/6848
Inventor 叶明亮由昕秦洪强
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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