Development Of Precision Targeted Metabolomics Method And Its Applications To Multiply Biological-Matriex Assay

Metabolomics is a novel omics technology as genomics, transcriptomics and proteomics at postgenome era. It can high-sensitivity and resolution snapshot and capture the metabolic response of living organisms to genetic and environmental modifications and external stimuli by qualitative and quantitative assay of smallmolecule metabolites present in a diversity of biological matrixes. Currently, metabolomics has been widely applied to the niches such as pharmacology and toxicology, disease pathogenesis, clinical diagnosis, plant growth and development and other areas and acquired significant progression and achievements. Conventional metbolomics was designed to globally profiling the sum of small-molecule metabolites present in the selected biological matrix, by employing NMR, LC-QTOF MS or GC/MS, it was usually defined as untargeted metabolomics. However, untargeted metabolomics is incapable of high-sensitivity and –resolution detecting all the metabolies present in the defined biological samples in terms of uniform analytical parameters for the entire metabolome. This drawback exerts substantial resistance on the pursuit of precision qualification and quantitification with metabolomics innovation. Alternatively, the premise of breakthrough in, targeted metabolomics has the capacity to achieve the robust and precision metabolome assay although the throughput renders somewhat limitation, while it is principly used to optimize individualized analytical parameters for each metabolite. Herein, this study was designed to optimize and develop a hightthroughput precision-targeted metabolomics method with relatively broad coverage to over 200 metabolites with important bioactivity, and enable it to good enough for the precision metabolome assay of a variety of biological matrices.In this study, we were first to determine TQ MS based MRM analytical parameters for 203 metabolites with important biological function including sugars, amino acids, nucleic acids, vitamins, organic acids and alkaloids by the combinational mode of manual and automatic optimization. Next, LC analytical parameters were optimized as well to couple to the mass spectrometry assay, as the chemistry and temperature of column, the composition, gradient program and p H value of mobile phase were thoroughly optimized to allow this LC/MS method to maximally determine those key metabolites. As an expected result, 152 of 203 metabolites can be high-robust and sensitivity determined by this optimized LC/TQ MS method. Furthermore, to investigate the validation and feasibility of this LC/MS based preceision targeted metabolomics method, limit of detection(LOD), limit of quantification(LOQ), linearity, repreatability and stability were assessed and verified. Respresentive compounds fructose, cytosine, leucine, niacin, thiamin and acetylcholine were selected for the asaay of linearity and inter-/intra-day precision, as our results demonstrated that all the correlation coefficient(R2) of linear regression equation for those representative compounds is greater than 0.999, and the relative standard deviation(RSD) of the intraand inter-day precision are less than 5%. In short, such assessment verified the high sensitivity, stability and reproducibility of the developed precision-targeted metabolomics method, suggesting the capacity for the broad-scale metabolomics assay.In addition, to determine the applicability of the method, it has been broadly employed to investigate the targeted metabolome of different biological matrixes, involving body fluid(urine and plasma), bacterial and mammalian cells, plant extract and insect tissue. Then, the defined pattern recognition methods such as heatmap, PCA were used to analyze the metabolome data between control group and treated group. Our data revealed that this method well capture the defined metabolome produced by different biological matriexes whatever they are body fluid, plant extract, insect tissue or even microbial and/or mammalian cells, and the group classification revealed this method rendered high-reproducibility and- differeniability again.Take altogether, this developed targeted metabolomics method has the capability to relatively high-throughput,-sensitivity and-resolution determination of the defined metabolome present in a diversity of biological matrixes, it not only provided novel insight into the optimization and development of precision targeted metabolomics method but also further promised to investigate broadly biological questions proposed by different niches from metabolic perspective.