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Method for detecting adenosine deaminase activity through liquid chromatography tandem mass spectrometry and screening inhibitor thereof

A technology of adenosine deaminase and tandem mass spectrometry, which is applied in the field of determination of adenosine deaminase activity and screening of its inhibitors by liquid chromatography tandem mass spectrometry, achieving the effects of high sensitivity, low cost and simple operation

Inactive Publication Date: 2018-07-24
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] However, there is no liquid chromatography tandem mass spectrometry method for the determination of adenosine deaminase activity and screening of adenosine deaminase inhibitors with good precision, high accuracy, and strong stability that can meet clinical requirements.

Method used

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  • Method for detecting adenosine deaminase activity through liquid chromatography tandem mass spectrometry and screening inhibitor thereof
  • Method for detecting adenosine deaminase activity through liquid chromatography tandem mass spectrometry and screening inhibitor thereof
  • Method for detecting adenosine deaminase activity through liquid chromatography tandem mass spectrometry and screening inhibitor thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] 1. Experimental method

[0077] 1 Instruments and reagents

[0078] 1.1 Instrument

[0079] Vortex instrument (Votex-Genie 2, USA), BT-25S electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd.). KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). Micropipette (Eppendorf, USA). Milli-Q water purifier (Milli-Q, Millipore Co., Billerica, MA, USA). Microplate constant temperature shaker (Hangzhou Aosheng Instrument Co., Ltd.).

[0080] 1.2 Reagents

[0081] Adenosine deaminase (ADA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Adenosine (AD), inosine (Hypoxanthine ribonucleoside), chlormequat (internal standard, IS), were purchased from Sigma-Aldrich (MO, USA). EHNA and 2'-deoxycometamycin (2'-dCF) were purchased from Dalian Meilun Biotechnology Co., Ltd. Chromatographic grade methanol was purchased from Fisher Co. (NJ, USA). Chromatography grade formic acid was purchased from Tedia Inc. (OH, USA). Ul...

Embodiment 2

[0126] (1) Obtain whole blood from the orbital venous plexus of SD rats with heparin anticoagulation, and non-anticoagulant centrifugation to obtain serum and blood cells, dilute with pH 7.5 11mM PBS buffer and determine the protein concentration to obtain a protein concentration of 18mg / mL The sample to be tested; add the substrate adenosine to the sample to be tested, the final concentration of adenosine is 3.740 μM, react at 30°C for 22min, immediately stop the reaction with ice methanol at 0°C, take the reaction termination solution, add 3.5 Double the volume of water to dilute, mix, and centrifuge to obtain the supernatant of the reaction termination solution.

[0127] (2) According to the method of Example 1, the inosine content in the supernatant of the reaction termination liquid is determined by liquid chromatography tandem mass spectrometry:

[0128] The liquid phase conditions are as follows: Chromatographic column: 1.8 μm ACQUITY UPLC HSS T3 chromatographic column;...

Embodiment 3

[0132] (1) Take blood from the orbital venous plexus of SD rats to obtain whole blood with heparin anticoagulation, and non-anticoagulant centrifugation to obtain serum and blood cells, dilute with 9mM PBS buffer solution with pH 7.7 and determine the protein concentration to obtain a protein concentration of 22mg / mL The sample to be tested; add the substrate adenosine to the sample to be tested, the final concentration of adenosine is 3.750μM, react at 40°C for 18min, immediately stop the reaction with ice methanol at 0°C, take the reaction termination solution, add 4.5 Double the volume of water to dilute, mix, and centrifuge to obtain the supernatant of the reaction termination solution.

[0133] (2) According to the method of Example 1, the inosine content in the supernatant of the reaction termination liquid is determined by liquid chromatography tandem mass spectrometry:

[0134] The liquid phase conditions are as follows: Chromatographic column: 1.8 μm ACQUITY UPLC HSS ...

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Abstract

The invention relates to a method for detecting adenosine deaminase activity through liquid chromatography tandem mass spectrometry and screening an inhibitor thereof. The method comprises the following steps: reacting a sample (whole blood, serum or hemocyte) to be detected and substrate adenosine, or reacting the sample (traditional Chinese medicine or compound) to be detected, an adenosine deaminase and substrate adenosine; then detecting inosine content in the stop solution by the liquid chromatography tandem mass spectrometry; and calculating the activity of adenosine deaminase. The method has the advantages of being high in accuracy, high in precision, high in sensitivity, high in stability, simple to operate, low in cost, and high in throughput; an effective method is provided to clinically detect the adenosine deaminase activity and diagnose leukemia, typhoid fever, systemic lupus erythematosus, diabetes, hepatopathy and tumor, and the support is provided to screen the inhibitor of the adenosine deaminase in order to treat the abovementioned diseases.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a method for measuring the activity of adenosine deaminase by liquid chromatography tandem mass spectrometry and screening its inhibitors. Background technique [0002] Adenosine deaminase (Adenosine deaminase; Adenosine aminohydrolase; ADA; EC3.5.4.4) is a sulfhydryl enzyme that has an important relationship with the body's cellular immune activity, and each molecule contains at least 2 active sulfhydryl groups. It is also a key enzyme in the metabolism of purine nucleotides, which can specifically catalyze the deamination of adenine nucleoside or deoxyadenosine nucleoside to generate inosine nucleoside or inosine deoxynucleoside, which is then catalyzed by nucleoside phosphorylase Hypoxanthine is eventually oxidized to uric acid and excreted from the body. [0003] ADA is widely distributed in multiple tissues of the human body, especially in the mesentery, liver...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/045
Inventor 王长虹刘伟程雪梅戚胜兰管辉达
Owner SHANGHAI UNIV OF T C M
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