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Human serum O-glycosylation identification method based on chemical enzymatic catalysis

An identification method and glycosylation technology, applied in the direction of scientific instruments, material inspection products, measuring devices, etc., can solve the problems of low identification efficiency and inability to meet large-scale screening of disease markers, so as to improve identification reliability and specificity High performance and good spectral quality

Inactive Publication Date: 2020-05-15
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

Obviously, this identification efficiency is very low and cannot meet the requirements of large-scale screening of disease markers. A new high-sensitivity analysis method for O-linked glycosylation in human serum is urgently needed

Method used

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  • Human serum O-glycosylation identification method based on chemical enzymatic catalysis
  • Human serum O-glycosylation identification method based on chemical enzymatic catalysis
  • Human serum O-glycosylation identification method based on chemical enzymatic catalysis

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Experimental program
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Effect test

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 DTT with a final concentration of 20mM, 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 50mM sodium phosphate solution (pH=7.5), add PNGase F w...

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Abstract

The invention relates to a method for detecting mucoprotein type O-linked glycosylation (O-GalNAc type). The method is based on a strategy of combining biological enzyme selective oxidation and biological orthogonal chemical reaction; alactose 6-hydroxyl is oxidized into an aldehyde group by utilizing galactose oxidase, the glycopeptides are specifically captured and released through hydrazide chemistry, the O-GalNAc glycoform high sensitivity analysis is achieved by using the mass spectrometry-based detection method, and 59 O-GalNAc glycosylation modification sequences can be detected from 50[mu] L of human serum, and can correspondingly exceed 30 O glycated proteins. The method has the characteristics of high detection sensitivity, high enrichment specificity and the like, and is an important means for analyzing the existing endogenous O-GalNAc type glycosylated protein / peptide fragment.

Description

technical field [0001] The invention belongs to the O-linked glycosylation proteomics analysis in the direction of proteomics research, and specifically relates to a chemical enzymatic method and liquid chromatography-mass spectrometry technology for the analysis of O-linked glycosylated proteins in human serum Enrichment and identification, this chemo-enzymatic method can achieve highly specific enrichment and highly sensitive identification of O-linked glycosylated proteins in serum. Background technique [0002] Human serum has long been the focus of basic and clinical research because of its ability to reflect the level of human health (literature 1. Maurya, P.; Meleady, P.; Dowling, P.; Clynes, M. Anticancer research Proteomic approaches for serum biomarker discovery in cancer 2007, 27, 1247-1255.). In order to discover more potential disease markers from serum, scientists have considered many new proteomic analysis methods based on mass spectrometry (2. Geyer, P.E.; K...

Claims

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Application Information

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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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