| The metabonomics is an emerging subject which have developed in 90's later period. It's to stimulates the change of the endogenous metabolite through the life individual by the disease physiology or the heredity decorate, and to study the whole biology condition. The metabonomics's object mainly is the biological body fluid, like the blood sample, the urine sample and so on, the attention are small member metabolites whose molecular weight is smaller than 1,000 ,the research method mainly is NMR and MS. For several years, the people have done the massive research to the nucleic acid and the protein through the genomics,the transcriptomics and the proteomics, however the small member metabolite was also noticeable, which were the windows of physiology pathological state. Metabonomics is to reflect the organism's physiological pathology condition through the study of the content and structure change of endogenous metabolite, then to study extraneous source matter effect through the relates with other"omics"technology.The metabonomics although is an emerging subject, but it was already became the important construction of system biology domain after the genomics,the transcriptomics and the proteomics, and was becoming the hot spot day by day in the last few years. At present, metabonomics study is already widely applied to the toxicology and the pharmacology, the function genomics, the disease diagnoses, and so on plant physiology, nutriology, as well as chinese traditional medicine modernization research.The kidney is the important excretory organ of human body which may drain vivo metabolite,the medicine, the poison and adjust body water, electrolyte, acid and alkali balance.When nephrosis has been destroyed by the harm from the disease and the injurious substance for a longer time,nephrosis cannot fully discharge the waste metabolites, thus many waste metabolites may remain in the body and the electrolyte and the balance between acid and alkali are disordered. This is chronic kidney disease(CKD).The CKD which develops to the end stage can cause the kidney essence harm, which lead to uremia. Once uremia happen, there are only two kinds of methods to treat and cure what are the dialysis regimen and the kidney transplant.But these two methods are expensive, dangerous and the success ratio was low. Therefore, the earlytime discovered and obtains the prompt treatment and has the count formuch significance after the patient survival.In this research, we have established the metabonomics analytical method in serum with LC-MS utilizing the metabonomics technique, and applied to the nephrosis metabonomics study. we have conducted the exploration research of the nephrosis pathology change through the contrast between the nephrosis serum and the normal serum metabolite and we hope to establish the nephrosis metabonomics model which may assist us to seek the possible biomarker.This research has laid the foundation for early diagnosis of the nephrosis. This research is divided into two parts: First part, the establishment of metabonomics anyalytical method with LC-MS in serum;Second part, the application in analysis of nephrosis.First part, the establishment of metabonomics anyalytical method with LC-MS in serum: We have carried on the protein precipitation and extracted the endogenous metabolite from the serum by CH3OH.we divide LC-MS into two parts to inspect: chromatographic fractionation and MS analysis in order to obtain as far as possible many metabolites. first, we have made the optimization for some parameters of MS which have importance contribution for ionization efficiency:Capillary voltage, Cone voltage and source temperature and so on. Second, we have selected three kinds of chromatographic columns and inspected their separation performance;we have inspected the separation effect of three kinds of mobile phases(H2O-CH3OH, 0.1% formic acid and 0.05% formic acid)under ESI+; we have also inspected the separation effect of three kinds of mobile phases(H2O-CH3OH,20mM NH4Ac and 10mM NH4Ac)under ESI-.At last,we determined the best MS and HPLC condition After the comprehensive survey and has established a feasible metabonomics analytical method with LC-MS in serum which has laid the foundation for the analysis of nephrosis.Second part, the application in analysis of nephrosis: 40 examples(20 normal serums and 20 nephrosis serums) are detected by LC-MS and primitive data is gained.The primary data is pretreated by the Micromass Markerlynx software.Then the data is analyzed by pattern recognition method which is composed of PCA and PLS-DA in Markerlynx and SIMCA.The result is exported by the score chart and the loading chart. The result show that two groups have been separated obviously and have no any overlapping. The metabolite which have made the contribution for the separates are analyzed further by FT-MS and we obtained its precise molecular weight.According to the above its possible molecular formula and the structure have inferred.The result show that the FT-MS examination result is obviously consistent with the LC-MS result, the error does not surpass 0.01ppm. Moreover, for some metabolites with the bigger content changes between two groups, we have also carried on the preliminary analysis, and has laid the foundation for the next step of structure determined.To sum up , we have taked spraying mass spectra as the check instrument which is united ESI+ and ESI- scans, and selected the phenyl chromatographic column ,adopted the gradient method to wash the metabolites , then has built the metabonomics method adapted to the person serum and has realized first step studying of the nephrosis serum, and has got ideal effect.The research has laid the foundation for the early diagnose of nephrosis and the application of LC-MS in clinically study.
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