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A method for screening target protein ligands from organism metabolites

A technology of metabolites and organisms, applied in the fields of analytical chemistry and biochemistry, can solve the problems of low analytical throughput, infiltration of omics thinking, SEC resolution and backward development of omics, etc., to achieve simple processing procedures and improve screening Effects, effects of reliable high-throughput research tools

Active Publication Date: 2021-10-15
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

After incubating the protein with the small molecule mixture, Muckenschnabel et al. used SEC to remove unbound small molecules and performed LC-MS analysis on the protein fragments to characterize the small molecules that interacted with the protein, but limited by the limitations of SEC at that time. The resolution and the development of omics are lagging behind, and the accuracy and throughput of the analysis results need to be improved
Krylov et al. developed a dynamic size exclusion method and applied it to the kinetic study of small molecule-protein interactions (1 to 1 system), which has a good analytical effect, but its analytical throughput is low and has not Infiltrate omics thinking into it

Method used

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  • A method for screening target protein ligands from organism metabolites
  • A method for screening target protein ligands from organism metabolites
  • A method for screening target protein ligands from organism metabolites

Examples

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Embodiment 1

[0022] Example 1. Screening of carbonic anhydrase endogenous ligands from biological metabolites based on dynamic size exclusion chromatography-mass spectrometry.

[0023] In the first step, 5 μL of the spiked serum metabolite extract (with the addition of acetazolamide, a known ligand of carbonic anhydrase) and 5 μL of the blank mobile phase were sequentially loaded onto the size exclusion chromatography column for separation using interval injection mode. And use mass spectrometry for online analysis;

[0024] In the second step, 5 μL of spiked serum metabolite extract (acetazolamide, a known ligand of carbonic anhydrase added) and 5 μL of carbonic anhydrase at a concentration of 5 μM were successively loaded onto the size exclusion chromatography column by interval injection mode. Separation, and use mass spectrometry to carry out on-line analysis; Fix the loading amount of the serum metabolite extract to make it the same as step (1);

[0025] In the third step, 5 μL of sp...

Embodiment 2

[0033] Example 2. Screening of endogenous ligands of human serum albumin from biological metabolites based on dynamic size exclusion chromatography-mass spectrometry.

[0034] In the first step, 5 μL of serum metabolite extract and 5 μL of blank mobile phase were sequentially loaded onto the size exclusion chromatography column for separation using interval injection mode, and online analysis was performed by mass spectrometry;

[0035] In the second step, 5 μL of serum metabolite extract and 5 μL of human serum albumin with a concentration of 5 μM were successively loaded onto a size exclusion chromatography column for separation using interval injection mode, and online analysis was performed by mass spectrometry; fixed spiked serum metabolism Extract load sample size makes it identical with step (1);

[0036] In the third step, 5 μL of serum metabolite extract and 5 μL of human serum albumin at a concentration of 25 μM were sequentially loaded onto a size exclusion chromato...

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Abstract

The invention discloses a method for screening endogenous ligands of target proteins from biological metabolites based on dynamic size exclusion chromatography (KSEC)-mass spectrometry. The metabolite extract and the target protein are sequentially loaded into the size exclusion chromatography column by interval injection mode. Since the molecular size of the protein is much larger than the metabolite, it has a faster migration speed in the column, so the target protein will pass through the metabolite extraction. The product fraction elutes from the column first. By fixing the concentration of the metabolite extract and continuously increasing the concentration of the target protein, high-throughput screening of metabolites that interact with the target protein can be performed from the metabolite extract by means of non-targeted metabolomics analysis. This method does not require additional labeling or immobilization of proteins or metabolites, and the direct series connection of size exclusion chromatography and mass spectrometry makes the pretreatment process simpler, and the whole process does not use organic reagents to improve the detection of hydrophilic ligand metabolites. The screening effect is a reliable high-throughput research method for metabolite-protein interactions.

Description

technical field [0001] The invention relates to the fields of analytical chemistry and biochemistry, and is a method for screening endogenous ligands of target proteins from biological metabolites based on dynamic size exclusion chromatography-mass spectrometry technology. Background technique [0002] In organisms, in addition to serving as intermediates in the metabolic transformation process, metabolites also serve as response signals to directly or indirectly trigger adaptive responses, participating in such as lipid synthesis, glycolysis pathway, lipid breakdown, proteolysis, three Physiological processes such as the carboxylic acid cycle. Cell nutritional status, external stress, and ecological conditions affect the levels of thousands of metabolites in cells, and these molecularly mediated signals are transmitted through a series of molecular events including metabolite-protein functional interactions . Therefore, an in-depth study of the interaction between endogen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/18G01N30/72
CPCG01N30/02G01N30/18G01N30/72
Inventor 许国旺王博弘石先哲秦倩
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI