Method for screening and characterizing metabolite-protein interaction system

Abstract

The invention discloses a method for screening and characterizing a metabolite-protein interaction system based on metabolomics and a structural mass spectrometric technique; a heterologously expressed tagged target protein is immobilized through an affinity immobilization method, then, the target protein is incubated with a metabolite extract, and finally, after being separated, denatured and eluted, the target protein-metabolite complex is subjected to non-targeted metabolomics analysis, and the complex is compared with a control group (an affinity vector without immobilized protein) so as to screen a metabolite with potential interaction with the target protein. Afterwards, the potential interaction system is directly verified using structural mass spectrometry. The binding method enables high-throughput screening of metabolites with potential interaction with the target protein, and also, direct characterization of the structural mass spectrometry can be used to screen out false positive interaction results that are probably introduced during indirect characterization. The method is a reliable high-throughput research tool for protein-metabolite interactions.

Description

technical field [0001] The invention relates to the fields of analytical chemistry and biochemistry, and is a high-throughput screening and characterization method for metabolite-protein interaction systems that combines indirect screening of metabolomics and structural mass spectrometry to directly characterize the two technical routes. Background technique [0002] Small endogenous metabolite molecules are the main components of intracellular molecular compounds, and various metabolite molecules have large differences in intracellular concentration distribution. The interactions it involves with a wide variety of proteins (whether by participating in enzymatic reaction processes in the form of products / substrates or allosterically regulating proteins in the form of allosteric cofactors / ligands) have been It proves that it can control the material and energy metabolism in biochemical reactions and can regulate the entire physiological process through signal transduction. T...

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Problems solved by technology

Saghatelian et al. expressed the target protein heterologously in vitro, and used metabolomics to screen the metabolites that may interact with the protein, but there is a possibility of non-specific adsorption during the material adsorption process, which may introduce false positive results; Klassen et al. Structural mass spectrometry can directly characterize the interaction between small molecules and proteins, and can obtain related thermodynamic and kinetic parameters, but this method cannot screen large quantities of metabolites that interact with target proteins, and is only applicable A relatively simple research system

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