Comparative Study On Biosample Preparation And Data Pretreatment For NMR-based Metabolomics

Serving as two critical steps in metabolomics research,biosample preparation and data processing can affect the reliability and accuracy of analytical results to a great extent.In this thesis,the preparation of two commonly used biological samples of serum and feces and pretreatment of 1H NMR spectral data based on NMR metabolomics were compared and researched.The main contents are presented as follows:The first part presented the effects of serum sample preparation methods on subsequent analysis results.In order to decrease the effects of macromolecules like proteins and lipids in sample on 1H NMR metabolic profile of serum,some researchers recommended that the macromolecules in serum should be filtered before NMR experiments and proposed some sample preparation methods.However,effects of sample preparation methods on the results of subsequent metabolomics analysis remained to be further researched.Aiming this problem,an experiment was designed to analyze 1H NMR spectra of three sample preparation methods including methanol extraction,acetonitrile extraction,and ultrafiltration tube filtration and compare these methods to conventional method in this thesis.The results presented that these three sample preparation methods both own the ability of removing macromolecules in 1H NMR spectra better,and the IH NMR spectra of methanol extraction method shown better SNR,resolution,reproducibility,metabolites retention and costs than other methods.The second part researched the impacts of fecal samples' storage conditions on subsequent analysis results.This experiment focused on the effects of storage time and storage temperature on 1H NMR metabolic profile of fecal sample.The storage time was set at three levels:24 hours,1 week and 1 month,and the storage temperature was set at two levels of-80 ? and-20 ?.The results of multivariate statistical analysis and two-way analysis of variance implied that effects of storage time on the metabolic profile was greater than the storage temperature.In NMR experiments,the storage time of fecal samples should not exceed one week.Fecal metabolic profiles can maintain better at the storage temperature of-80 ?,which would change within few hours at the storage temperature of-20 ?C.The third part researched the effects of pretreatment methods of 1H NMR metabolic profile on subsequent analysis results.Piecewise integral and(semi)quantitative analysis are the two main pretreatment methods for the 1H NMR metabolic profile.The SD rats' NMR data of moxibustion gallbladder meridian experiment,were used as research objects to compare two data pretreatment methods,quantitative analysis by SpectFitting software and conventional full-spectrum segmentation integral.The results shown that the segmentation integration method'analysis results appeared more comprehensive because of the usage of full-spectrum information,but semi-quantitative analysis method' results were more reliable.