Undiscriminated protein thermal stability analysis method

An analysis method and thermal stability technology, applied in the direction of analyzing materials, material inspection products, measuring devices, etc., can solve problems such as no revelation, achieve wide coverage, avoid experimental errors, and non-discriminatory effects

Pending Publication Date: 2021-06-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The above literatures do not make any record or enlightenment on the analysis method of protein thermal stability based on non-discrimination

Method used

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  • Undiscriminated protein thermal stability analysis method
  • Undiscriminated protein thermal stability analysis method
  • Undiscriminated protein thermal stability analysis method

Examples

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Embodiment 1

[0039] The new method of protein thermal stability analysis based on non-discrimination is used in the research of the target protein of Stauroporine (stauroporine):

[0040] (1) Take 1 plate of K562 cell samples, resuspend in 600 μL PBS buffer solution, add protease inhibitors (AEBSF, aprotinin, bestatin, E-64, leupeptin) with a final volume concentration of 1% dimethyl sulfoxide (DMSO) solution of a mixture of peptide and pepstatin A), after repeated freezing and thawing through liquid nitrogen three times, centrifuge at 20,000g for 10min at 4°C, and collect the supernatant sample;

[0041] (2) Take 50 μL of the above protein samples in eleven new EP tubes at 37°C, 41°C, 44°C, 47°C, 50°C, 53°C, 56°C, 59°C, 62°C, 65°C, Heat at 68°C for 3 minutes, then cool at room temperature for 2 minutes, and then centrifuge at 4°C and 20,000g centrifugal force for 10 minutes. Take 30 μL of the supernatant in each EP tube and transfer them to eleven new EP tubes for trypsin (trypsin) diges...

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Abstract

The invention relates to an undiscriminated protein thermal stability analysis method used for screening target protein interacting with ligand molecules. The method comprises the following steps: taking a protein sample, respectively adding and not adding ligand molecules to incubate, heating, separating supernate, carrying out enzymolysis and quantitative proteomics analysis, calculating the difference of protein quantitative results of a ligand group and a blank group, and screening out the ligand target protein. Experiments are only carried out at temperature points where the thermal stability of most of proteins is greatly changed, temperature points which are not meaningful to ligand molecule target protein identification are omitted, and the difference between the proteins of a ligand group and a blank group is directly calculated without calculating delta Tm through a fitting curve; and the difference of protein thermal stability of the target protein in the ligand adding group and the blank group is more visually reflected, the situation that many target proteins with irregular melting curves are omitted due to the fact that delta Tm cannot be calculated can be avoided, the method is suitable for all proteins responding to heat, and the coverage degree of target protein identification is improved.

Description

technical field [0001] The invention belongs to the technical field of protein-ligand molecular interaction in the direction of proteomics research, and specifically relates to a high-sensitivity and high-coverage ligand target protein identification method and its application. Background technique [0002] The comprehensive and accurate identification of target proteins and off-target proteins of drug molecules has always been a major challenge in the field of drug research. In recent years, with the rapid development of mass spectrometry technology, several mass spectrometry-based proteomics techniques have been applied to the identification of drug molecular target proteins. In addition to having higher throughput, these techniques can better identify direct and indirect target proteins of drug molecules without any modification of drug molecules. These include: drug affinity responsive target stability method (DARTS) (Target identification using drug affinity responsive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6848
Inventor 叶明亮阮成飞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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