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Method for rapid high-efficiency qualitative and quantitative analysis of N-sugar in virtue of high-performance liquid chromatography

A high-performance liquid chromatography and quantitative analysis technology, which is applied in the direction of analyzing materials, measuring devices, and material separation, can solve the problems of unsatisfactory sensitivity and long time consumption, so as to improve analysis efficiency and detection sensitivity, enhance analysis sensitivity, and respond The mild effect of the conditions

Inactive Publication Date: 2017-11-21
EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH +1
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] In the prior art, the N-glycan analysis steps are generally divided into: deglycosylation, sugar extraction, sugar labeling, purification, and sugar analysis. The traditional N-glycan analysis method uses PNGase F to digest and deglycosylate overnight, and then needs Purification of the released glycans, labeling of the N-glycan reducing ends by reductive amination, and further purification of the derivatized mixture before HPLC analysis often takes several days and consumes time is long and the sensitivity is not very ideal

Method used

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  • Method for rapid high-efficiency qualitative and quantitative analysis of N-sugar in virtue of high-performance liquid chromatography
  • Method for rapid high-efficiency qualitative and quantitative analysis of N-sugar in virtue of high-performance liquid chromatography
  • Method for rapid high-efficiency qualitative and quantitative analysis of N-sugar in virtue of high-performance liquid chromatography

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A fast and efficient N-sugar qualitative and quantitative analysis method utilizing high performance liquid chromatography, specifically comprising the following steps:

[0025] (1) Add 10 μg of glycoprotein RNase B, fetuin or 10 μL of human serum to ammonium bicarbonate buffer and denaturing buffer at pH 8.5. The total volume is 20 μL, of which the denaturing buffer is 0.48 μL. Denatured in a boiling water bath for 10 minutes, after cooling to room temperature, 2.4 μL of 10% NP-40 was added to inhibit the denaturant for 5 minutes.

[0026] (2) Add 1 μL of exoglycosidase PNGse F, microwave-assisted enzyme digestion for 20 min, in order to prevent the sample from boiling, the sample was floated in a beaker filled with 500 mL of water and reacted in a microwave oven with 700W power.

[0027] (3) After the reaction is completed, cool to room temperature, then directly add 20 μL of 150 mM AQC acetonitrile solution and 20 μL of pH 8.5 ammonium bicarbonate buffer, shake and m...

Embodiment 2

[0030] Glycoprotein RNase B was selected for condition optimization, fetuin was used for further verification, and protein SDS-PAGE was used to explore the effects of microwave time and the molar ratio of enzyme to substrate during PNGase F digestion, and compared with the traditional overnight digestion that can completely digest By comparison, the most suitable molar ratio of enzyme and substrate and microwave irradiation time were obtained. Specific steps are as follows:

[0031] (1) After 10 μg of RNase B is denatured in the manner of Example 1;

[0032] (2) Control the microwave radiation time to 20min, add 1:10000, 1:5000, 1:2500, 1:1000 molar ratio of enzyme to substrate for experiment, and carry out the experiment with the traditional overnight enzyme digestion that can completely digest Compared;

[0033] (3) Select the optimized molar ratio of enzyme and substrate in step (2) of 1:1000, and do a verification test on the complex glycoprotein fetuin, and find that th...

Embodiment 3

[0037] After RNase B digestion was completed using the optimized microwave-assisted digestion conditions, 20 μL of AQC solution and 20 μL of digestion reaction buffer were added, and the reaction was performed in a water bath.

[0038] Through the factors affecting AQC labeling reaction, the different types of reaction buffer (sodium phosphate buffer, ammonium bicarbonate buffer, sodium borate buffer) and concentration (10, 20, 50, 80, 100) mM, the concentration of derivative reagent (30, 50, 100, 150, 200) mM, reaction pH (7.5, 8.0, 8.5, 9.0), temperature (25, 40, 50, 60, 70) °C and reaction time (5, 10, 20, 40 , 60) min for exploration and optimization. The optimization experiment adopts a single factor analysis method, that is, one factor is selected for gradient analysis, and other factors are kept the same. After confirming the condition of one factor, the analysis of other factors is carried out similarly.

[0039] attached by figure 2 The optimal reaction condition ...

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Abstract

The invention provides a method for rapid high-efficiency qualitative and quantitative analysis of N-sugar in virtue of high-performance liquid chromatography, belonging to the field of glycomic analysis. According to the method for rapid high-efficiency qualitative and quantitative analysis of N-sugar in virtue of high-performance liquid chromatography, a microwave-aided means is employed for accelerating the deglycosylation reaction of N-sugar, and a derivative reagent AQC is cooperatively used for marking glucosamine directly obtained through enzyme digestion, so high-efficiency high-sensitivity qualitative and quantitative analysis of N-sugar is realized.

Description

technical field [0001] The invention belongs to the field of glycomics analysis, and in particular relates to a fast and efficient N-sugar qualitative and quantitative analysis method using high-performance liquid chromatography. Background technique [0002] N-glycosylation of proteins plays an important role in many important biological processes, such as cell signaling, fertilization, proliferation and differentiation. Alterations in protein N-glycans have been implicated in the pathogenesis of many diseases including cancer, diabetes and heart failure. Therefore, stable identification and accurate quantification of N-glycans are highly necessary for gaining further understanding of related biological processes and for discovering new diagnostic markers of glycometabolism and therapeutic drug targets. [0003] In the prior art, the N-glycan analysis steps are generally divided into: deglycosylation, sugar extraction, sugar labeling, purification, and sugar analysis. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/02
CPCG01N30/06G01N30/02G01N2030/067
Inventor 刘欣
Owner EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH
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