Detection Of Small Molecule Metabolites In Biological Samples

In recent years,metabolomics has played a key role in early disease diagnosis,drug target discovery,disease mechanism research,and disease diagnosis.Metabolomics is an emerging research method developed after genomics,transcriptomics,and proteomics.It is an indispensable part of a comprehensive understanding of biological systems.Metabolomics research targets all low molecular weights within a specific time.(Usually Mw <1500)The sum of metabolites,which focuses on the changes of the entire metabolite group.The key is to perform qualitative and quantitative analysis of all metabolites in the target system.The research of metabolomics has positive significance for human health,drug development,food field,traditional Chinese medicine research,environmental safety,and metabolic engineering.This project is mainly aimed at the research needs of targeted metabolomics,and has developed a method for detecting metabolites in the glycolysis process and organic acids in the tricarboxylic acid(TCA)cycle in common key metabolic pathways in organism metabolism.Taking E.coli as an example,the pretreatment method and derivatization of metabolites in biological samples were optimized.Gas Chromatography-tandem triple quadruple Mass Spectrometry(GC-MS / MS)and liquid phase were used.Liquid Chromatography-tandem triple quadruple Mass Spectrometry(LC-MS / MS)established a method for the detection of multiple metabolites in biological samples,and applied it to cancer cells,plant tissues and other organisms.Testing of samples.The main work of this subject is as follows:(1)The subject determined and compared the efficient extraction method of E.coli metabolites and the conditions for solid-phase extraction of organic acids in the TCA cycle,and established a complete pretreatment method for the detection of organic acids in the TCA cycle of E.coli.In the experiments,liquid nitrogen freeze-thaw and ultrasonic disruption methods were respectively used to break cell walls and extract metabolites.By comparing the amount of metabolites extracted,it was found that the liquid nitrogen freeze-thaw method can accurately extract cell metabolites and maintain their stability;The solid phase extraction method was used to remove the excess matrix interferences from the E.coli extract,and the solid phase extraction conditions were optimized so that the target compounds could be effectively extracted and the complex matrices such as proteins could be removed to avoid interference with the mass spectrometry detection;The selection of the organic acids and the optimization of the derivatization time and temperature make the six organic acids have better derivatization efficiency.The developed pretreatment method can efficiently extract and purify the targetdetection substance in the cell.The method has low detection limit and good reproducibility,and is suitable for the extraction and purification of metabolites in biological samples.(2)Corresponding GC/MS detection methods were developed for six organic acid derivatives.The separation of six organic acids on the end of the gas chromatograph was completed by the programmed temperature setting.Through optimization of the parent ion,the product ion,and the collision voltage,the mass reaction conditions of the multiple reaction monitoring mode(MRM)were established.The external standard method was used to quantify and detect the detection method.Methodological validation was performed.The six organic acids have good linearity in the range of 20 ?g/L-1×104 ?g/L,the correlation coefficients are all greater than 0.99,and the standard recovery is 82.3%-100.4%,and the relative standard deviation is 2.3%-4.5%.The output limit is 2.0 ?g/L-18 ?g/L.The method is less affected by the matrix and has good reproducibility,which is suitable for the determination of organic acid content in complex biological matrix samples.(3)The organic acid content in cancer cells and plant tissues was determined using the established method.Six types of organic acids in the samples could be detected.(4)After the organic acid detection method in E.coli has been established,the detection and analysis of glycolytic metabolites in E.coli are studied.First,the E.coli standard solution was configured,and the LC-MS/MS method of glycolytic metabolites was explored.The elution conditions and flow rate of acetonitrile and 10 Mm/L ammonium acetate were determined as 9:1.It was 300 ?L/min and the injection volume was 10 ?L.Successfully detected dihydroxyacetone phosphate(PEP),glucose(Glu),pyruvate,glucose-6-phosphate(G6P)/ fructose-6-phosphate(F6P),and fructose-1,6-diphosphate(FBP).Some glycolytic metabolites such as glucose-6-phosphate(G6P),fructose-6-phosphate(F6P),and3-phosphoglycerate(3-PG)have tried many methods including changing the column and optimizing the mobile phase.Later,it was still not well separated on the liquid chromatography,so GC-MS combined with derivatization was used for analysis and detection.MSTFA was used as the derivatization reagent to derivatize the standard of glycolytic metabolites.The injection analysis succeeded in detecting these substances,and the pyruvate and glucose were successfully detected in the actual sample E.coli test.