| A high-throughput metabonomics analysis method has been implemented by using rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS/MS) with intensive validation of endogenous metabolites’stability and precision. Untargeted and targeted analysis methods based on RRLC-MS/MS have been applied to investigate a large scale of lung cancer subjects and many reliable potential biomarkers for diagnosis have been found.Based on the previous study of plasma metabonomics in our laboratory, a high-throughput metabonomics analysis method has been implemented by using RRLC-MS/MS. Further the RRLC-MS/MS analysis method in both positive and negative ion modes combined with multivariate statistics was employed to investigate the short-term stability of metabolites in blood and plasma at different temperatures, as well as the long-term stability of metabolites in plasma delayed at-80℃for5years to summarize the change rules of metabolites systematically. The thorough understanding of the effects of metabolites’stability is not only necessary for improving the reliability of potential biomarkers, but also useful to provide pre-analytical operation guidance to both untargeted and targeted metabonomics. Untargeted LC-MS/MS analysis method established previously was applied to investigate lung cancer subjects to find biochemically significant metabolite biomarkers. After a large scale plasma samples were studied,74endogenous metabolites showing significant differences between lung cancer patients and healthy volunteers were found. Then a targeted LC-MS/MS metabonomics method, based on the74endogenous metabolites referred before, was developed and applied to validate the diagnosis biomarkers of lung cancer. By analyzing other batch plasma samples of lung cancer,38potential biomarkers related to the diagnosis of lung cancer were selected. Subsequently, high resolution MS and MS/MS analyses, as well as inter-metabolite correlation analysis were performed for the identification of the metabolites of interest. Until now,32potential biomarkers have been identified. The further metabonomics pathway analysis indicated that lung cancer was related to fatty acid metabolism.1. Untargeted metabonomics analysis method based on LC-MS/MS technologyOur laboratory has developed the method of plasma metabonomics based on LC-MS/MS in previous study. And on this basis a untargeted LC-MS/MS-based metabonomics using electrospray ionization (ESI) in both positive and negative ion modes has been further optimized in the field of pretreatment method of plasma samples and the analytical condition of liquid chromatography and mass spectrometry analysis, especially the re-dissolution solvents. The method has also been intensively validated for its precision, sensitivity and stability, ensuring the method is appropriate for untargeted plasma metabonomics study. The different chromatographic behavior was observed when the dried plasma samples were redissolved with different re-dissolution solvents, which affected chromatographic behavior and made some potential biomarkers identified by mistake. Systematic analysis of the effect of re-dissolution solvent on chromatographic behavior confirmed that initial mobile phase was the optimal re-dissolution solvent to acquire accurate data of metabolites. In addition, the methodology of this method was investigated and the results demonstrated that this method had high precision, good sensitivity, and could be used for the metabolomics analysis of plasma. Therefore, an accurate untargeted metabonomics based on LC-MS using ESI in both positive and negative ion modes was developed to analyze plasma samples.2. Investigation on the stability of plasma metabolitesThe short-term stability of metabolites in plasma at4℃or37℃after a delay in sample processing (0,1,2,4,8,12, and24h) and the long-term stability of metabolites in plasma at-80℃for five years were investigated by RRLC-MS/MS-based metabonomics analysis in positive and negative ion modes combined with multivariate statistics. Principal components analysis (PCA) and ten kinds of identified metabolites were used to summarize the time-dependent change rules in metabolites systematically at different temperatures in short-term stability study. The long-term stability of metabolites in plasma specimens stored at-80℃for five years was studied by OPLS-DA. Analysis of these subjects identified36metabolites with statistically significant changes in expression and found a kind of metabolites with hundred-fold change. These studies show that a thorough understanding of the effects of metabolite stability is necessary for improving the reliability of potential biomarkers. Meanwhile, it is useful to provide pre-analytical operation guidance to both untargeted and targeted metabonomics.3. Untargeted metabonomics study on lung cancerA metabonomics strategy method of untargeted plasma metabonomics based on LC-MS/MS has been applied to find biologically significant metabolite biomarkers in lung cancer. A large number of biological samples including232lung cancer patients and155healthy volunteers were analyzed to obtain abundant information and find the potential biomarkers that were closely associated with the diagnosis of lung cancer. Until now,74potential biomarkers have been found by RRLC-ESIMS in positive and negative ion modes in this metabonomics study, which demonstrates that these abnormal metabolites were related to metabolic disorders in lung cancer patients. Up to now50potential biomarkers have been identified by using high resolution MS and MS/MS analyses, as well as fragmentation rules of metabolites, which included18lysophosphatidylcholines,17acylcarnitines (8hydroxylacylcarnitines),7P-hydroxy fatty acids, et al. Twelve potential biomarkers have been identified by comparison with related standards by LC-MS/MS analysis.4. Targeted metabonomics study on lung cancer:validating potential diagnosis biomarkersIn present study, a targeted metabonomics based on74discriminating metabolites screened from untargeted metabonomics was developed using LC-MS/MS technology with electrospray ionization (ESI) in both positive and negative ion modes, and multivariate statistics. Another batch of biological samples including91lung cancer patients and66healthy volunteers were analyzed to validate the accuracy of discriminating metabolites and found more reliable potential biomarkers that were closely associated with the diagnosis of lung cancer. Analysis of these subjects found38metabolites with statistically significant changes in expression, and the identified32metabolites including17acylcarnitines (8hydroxylacylcarnitines),7β-hydroxy fatty acids,1lysophosphatidylcholines, et al. The level of17acylcarnitines and7β-hydroxy fatty acids was significantly lower in lung cancer plasma. Then the metabolic correlation and pathway analysis have been studied, suggesting lung cancer may be closely related with fatty acid metabolism. |
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