Quantitative detection method of O-GlcNAc

A quantitative detection method, o-glcnac technology, applied in the medical field, can solve problems such as low sensitivity, interference, need for labeling and enrichment, etc., and achieve the effects of avoiding interference, accurate quantitative detection, and simple operation

Inactive Publication Date: 2017-05-10
UNIVERSITY OF CHINESE ACADEMY OF SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a quantitative detection method for O-GlcNAc, aiming to solve the problem that the methods for detecting O-GlcNAc are mostly used in qualitative and semi-quantitative analysis, and there is interference from other GlcNAc sugars at the end, low sensitivity, complicated and expensive sample preparation , Difficult data processing, need for labeling and enrichment, etc.

Method used

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  • Quantitative detection method of O-GlcNAc
  • Quantitative detection method of O-GlcNAc
  • Quantitative detection method of O-GlcNAc

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Embodiment 1

[0030] First, the thiol acid was modified on the gold membrane through the interaction between gold and sulfhydryl groups, and then the standard peptides glycosylated with different concentrations of O-GlcNAc were modified on the gold membrane through the aminocarboxyl cross-linking reaction. , using the Navi-200SPR instrument to monitor the interaction between WGA and O-GlcNAc online in real time.

Embodiment 2

[0032] Since WGA can not only recognize O-GlcNAc, but also recognize other glycans with GlcNAc at the end, in order to avoid the interference of the GlcNAc at the end, the present invention adopts enzyme cutting technology. Firstly, mercaptoacids were modified on the gold membrane, and then O-GlcNAc glycosylated standard polypeptides of different concentrations were modified by aminocarboxyl cross-linking reaction, and then digested at 37°C for 16 hours, and then flow-injected nano-gold-labeled WGA, The signal was recorded by Navi-200 SPR instrument, and the working curve for detecting O-GlcNAc was established according to the change ΔR of the signal before and after enzyme digestion and the concentration of O-GlcNAc. The corresponding SPR signal and working curve are attached figure 2 ~ attached Figure 4 shown.

[0033] figure 2 It is the signal diagram of the SPR of WGA / AuNPs flow-injected with O-GlcNAc glycosylated standard polypeptides of different concentrations pro...

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Abstract

The invention discloses a quantitative detection method of O-GlcNAc, comprising the following steps: detecting O-GlcNAc according to SPR signals recorded according to change of the refractive index caused by the inter combination effect of wheat germ agglutinin WGA and O-GlcNAc on a gold film; cutting O-GlcNAcase (OGA) by using the specificity of O-GlcNAc, wherein signal decrease after cutting is related to the concentration of O-GlcNAc; and according to change of signal delta R before and after enzyme digestion, realizing accurate and quantitative detection of O-GlcNAc. The quantitative detection method of O-GlcNAc has low requirements on samples which can be detected after simple treatment, is simple in operation, has a wide linear range relative to the previously reported work, and can be applied to detection of O-GlcNAc in cancer cells.

Description

technical field [0001] The invention belongs to the field of medical technology, in particular to a method for quantitatively detecting O-GlcNAc. Background technique [0002] Oxygen-linked N-acetylglucosamine (O-GlcNAc for short) is composed of a single N-acetylglucosamine (GlcNAc, monosaccharide) with an O-glycosidic bond to the ser / threonine (Ser / Thr) residue of the protein part. The hydroxyl oxygen atom of the group is covalently bonded. O-GlcNAc modification is a ubiquitous protein post-translational modification. A large number of studies have proved that the level of O-GlcNAc modification is closely related to neurodegenerative diseases, diabetes, cancer and other diseases. However, the modified glycosidic bond of O-GlcNAc is easy to break, is at the substoichiometric level, has dynamic properties, and most of the proteins are low-abundance proteins, which brings inconvenience to the establishment of detection methods, especially for accurate quantitative detection. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/552G01N21/41G01N33/68
CPCG01N21/553G01N21/41G01N33/6893G01N33/6896G01N2800/042G01N2800/28G01N2800/7028
Inventor 姚鑫高利
Owner UNIVERSITY OF CHINESE ACADEMY OF SCIENCES
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