Electrochemical sensing detection method of protein O-GlcNAc glycosyltransferase activity

Abstract

The invention discloses an electrochemical sensing detection method of protein O-GlcNAc glycosyltransferase activity. The electrochemical sensing detection method comprises the following steps that a membrane of substrate polypeptide molecules of OGT is formed on the surface of an electrode; the obtained electrode fixed with the membrane of the polypeptide molecules on the surface and a glycosylation reaction fluid are incubated through OGT catalysis to obtain an electrode fixed with the membrane of the glycosylated polypeptide molecules on the surface; the electrode fixed with the membrane of the polypeptide molecules on the surface and the electrode fixed with the membrane of the glycosylated polypeptide molecules on the surface are sequentially subjected to the treatment in the steps 1-3 as follows: 1, the electrodes are put in a protease reaction fluid for hydrolysis; 2, the electrodes are cleaned with water; 3, a tyrosine residue serves as an electrochemical signal probe, and the electrodes perform electrochemical oxidation reaction; tyrosine residue electrocatalytic oxidized currents in the obtained systems are respectively determined, and OGT activity can be quantitively analyzed according to the obtained catalyzed and oxidized currents. By adopting the method, simple, quick and non-marked OGT activity detection is achieved through an electrochemical biosensor.

Description

technical field [0001] The invention relates to an electrochemical sensing detection method for protein O-GlcNAc glycosyltransferase activity, belonging to the field of biological analysis. Background technique [0002] Protein glycosylation is the most common and important covalent modification method in protein post-translational modification, and it is an important regulatory mechanism of protein function. Process (Wong C.H.J.Org.Chem., 2005, 70(11):4219-4225.). As a ubiquitous post-translational modification, glycosylation has an important impact on protein structure and function. Glycosylation is present in more than 50% of proteins in organisms (Apweiler R. et al. Biochim. Biophys. Acta, 1999, 1473(1): 4-8.), glycosylation is important for protein folding, transportation, Localization plays an important role (Helenius A.Science, 2001, 291(5512): 2364-2369.), almost all key molecules in the immune system are glycoproteins (Rudd P.M.et al.Science, 2001, 291(5512) :237...

AI Technical Summary

Problems solved by technology

The disadvantage is that the method involves chemical synthesis when the fluorescent probes at both ends of the polypeptide are labeled, the operation is complicated, and it is not universal.

In summary, although some protein glycosylation analysis and detection methods have been developed, there are still many defects in these methods, which cannot meet the actual requirements, and further research and development of new methods and new technologies are needed.

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