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Mass spectrum detection method for interaction between active protein and small molecule

A mass spectrometry detection and protein technology, which is applied in the field of interaction detection between active proteins and small molecules, can solve the problems of long analysis period, time-consuming and laborious, difficult purification, etc., and achieve the effect of long experiment period, cumbersome operation and improved analysis throughput

Active Publication Date: 2018-09-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods are time-consuming and labor-intensive, and the cycle of the analysis method is long. At the same time, many proteins, especially membrane proteins, are difficult to purify.

Method used

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  • Mass spectrum detection method for interaction between active protein and small molecule
  • Mass spectrum detection method for interaction between active protein and small molecule
  • Mass spectrum detection method for interaction between active protein and small molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Detection of Interaction between Catechol-O-methyltransferase (COMT) and Small Molecule Tolcapone (TCW)

[0040] Each of the purified catechol methyltransferases was dissolved in the following three reaction systems to keep the protein concentration at 1 mg / mL. The reaction systems were: 1) 50 mM phosphate buffered saline (PBS buffer), 1.6 mM Thiothreitol (dithiothreitol, DTT), pH 7.4; 2) 50mM PBS buffer, 1.6mM DTT, pH 7.4, 5mM MgCl 2 and 200μM S-adenosyl methionine (SAM); 3) 50mM PBS buffer, 1.6mM DTT, pH 7.4, 5mM MgCl 2 and 200 μM S-adenosyl methionine (SAM) and 10 μM TCW, and incubated on a temperature-controlled shaker at 25 degrees for 30 min. Add 10 mM C to the above three samples 13 D. 2 O and 15 mM NaCNBD 3 Carry out the dimethylation labeling reaction in the active state, the temperature is room temperature, and the reaction time is controlled at 30 minutes. After the reaction, 5 times the volume of protein precipitation solution and 50 mM ammonium acetate ...

Embodiment 2

[0044] Analysis of ATP-binding proteins in complex biological samples

[0045] Take 10,000,000 cells and disperse in 1mL active lysate, the active lysate components are 50mM Hepe buffer, 1mM EDTA, 150mM NaCl, 5% Glycerol, 1% NP-40, 1mM phenylmethylsulfonyl fluoride (PMSF). Under the condition of ice bath, ultrasonic-assisted cell disruption was performed, the ultrasonic power was 200W, the ultrasonic program was set to ultrasonic time 3S, interval time 5S, ultrasonic 30 times. Ultrasonic disrupted cells were subjected to high-speed centrifugation at 4°C to remove cell debris and other insoluble matter with a centrifugal force of 10,000 g and a centrifugation time of 10 min. The BCA protein concentration assay was used to determine the extracted protein concentration, and 6 parts of 200 μg extracted protein were taken respectively and divided into 2 groups (experimental group and control group), with 3 samples in each group. Dilute the protein concentration of each sample to ...

Embodiment 3

[0048] Interaction detection between bovine serum albumin (BSA) and 8-anilino-1-naphthalene sulfonate (ANS)

[0049] Each BSA was dissolved in the following three reaction systems to keep its protein concentration at 10mg / mL. The reaction systems were: 1) 100mM phosphate buffer (PBS buffer), 0.15mM ANS, pH 6.8; 2) 100mM PBS buffer , 0.6mM ANS, pH 6.8; 3) 100mM PBS buffer, pH 6.8, incubated on a 37-degree temperature-controlled shaker for 60min. Add 50mM C to the above three samples 13 D. 2 O and 50 mM NaCNBD 3Carry out the dimethylation labeling reaction in the active state, the temperature is 37 degrees, and the reaction time is controlled at 15 minutes. After the reaction, 4 times the volume of protein precipitation solution and 50 mM ammonium acetate were added, and the BSA protein was precipitated in a -20 degree refrigerator for 10 h. The precipitated protein was separated by high-speed centrifugation at 25000 g for 30 min at 4 degrees, and the precipitated protein was...

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Abstract

The invention provides a mass spectrum detection method for interaction between an active protein and a small molecule. According to the method, in-vivo covalent chemical labeling is performed on a protein while the active state is maintained; and the site and the intensity of the interaction between a small molecule and the protein are judged through the difference in the labeling efficiency before and after the interaction between the specific amino acid position on the protein and the small molecule. According to the present invention, with the mass spectrum detection method, the protein inhibitor candidate small molecule can be subjected to high-throughput screening, and the potential protein target of the specific small molecule can be subjected to large-scale identification analysis;and through the large-scale identification analysis of the Catechol-O-methyltransferase (COMT)-small molecule interaction and the ATP-binding protein in the complex system biological system, the method has accuracy and high efficiency.

Description

technical field [0001] The invention belongs to the technical field of detecting the interaction between active proteins and small molecules, and in particular relates to a mass spectrometry detection method for the interaction between active proteins and small molecules. Background technique [0002] During the growth and development of cells, proteins, as the main executors of life activities, interact with many endogenous or exogenous small molecules. By combining with small molecules, proteins can improve the stability of protein structure, change the three-dimensional structure of proteins and the execution and inhibition of protein biological functions. How to effectively detect the interaction between small molecules and proteins plays an important role in biochemical research and drug development. Through the large-scale analysis and detection of the interaction between small molecules and proteins, it is possible to effectively confirm the binding status and streng...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N33/68
CPCG01N30/88G01N33/6848G01N2030/8831
Inventor 王方军刘哲益周烨
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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