The Method And Application Of Chromatography-mass Spectrometry In The Analysis Of Microbial Metabolites

Metabolimics has been wildly used in microbial area and the expansion of its application is close related to the analytical techniques, especially the hyphenated techniques. Becouse of the combination of separation and analysis, chromatography-mass spectrometry methods are becoming one of the most powerful analysis systems.E.coli1.1566and△sdhAB、△ackA-pta gene knockout mutants were selected to this study. In order to compare the genetic and environmental perturbation on the metabolism of Escherichia coli, a sensitive method to analyze the metabolic profiling of the wild-type and two genetic knockout strains of E. coli cultured in different carbon sources by gas chromatography time-of-flight mass spectrometry (GC-TOF MS) was developed. The employed method was validated by representative standards. The calibration curves for most of the standards were satisfactory with regression coefficients better than0.99, the corresponding recoveries varied from66.6%to100.9%. The obtained metabolomics data from E. coli were subjected to partial least square-discriminate analysis (PLS-DA), showing clear separation between wild and genetic modified strains cultured in different carbon sources. Clustered heat map of the intercellular metabolism generated by hierarchical cluster analysis (HCA) indicated that the influence of carbon sources was more significant than that of the genetic factors. Also, the alteration of intercellular succinic acid proved the expected effects of deleting sdhAB gene in our first attempt.Exiguobacterium aurantiacum Lp3-3is one kind of alkaliphilic microorganism and has favorable apability to produce lactic acid. In this study, a practical metabolomics method based gas chromatography-mass spectrometry (GC-MS) was developed and used to study environment perturbation on metabolism of Lp3-3. The GC/MS method was validated by four representative isotope standards. The calibration curves for all the standards were satisfactory with regression coefficients better than0.99, the spilked recoveries varied from86.9%to102.1%and RSD were all better thanl0%. Multivariable data analysis was applied to the acquired data and showed differentiation when Lp3-3cultured in different medium. Finally, the metabolites playing key roles in the differentiation were further identified and lactic acid was the most obviously changed metabolite.Comprehensive two-dimensional gas chromatography(GC×GC) is a novel multidimensional separation technique which plays an important role in analysis of complex mixtures and has more advantages than ID GC. In this study, GC×GC combing with PC A method was applied to analyze the intracellular metabolites of E.coli. It showed that phenotype was differentiated from each other obviously.