著作: [鄭 丞弼]/[谷口 貴子]/Naoki Tani/[谷口 寿章]/Dansyl-labeling reversed-phase liquid chromatography- and mass spectrometry-based metabolomic investigation of human urine sample/第54回日本生化学会中国・四国支部例会
(英) Dansyl-labeling reversed-phase liquid chromatography- and mass spectrometry-based metabolomic investigation of human urine sample
(英) Metabolomics is the systematic study of endogenous small-molecule metabolites, the chemical entities that are transformed during metabolism, within a biological system. Analysis of these key metabolites is one of the powerful tools for the detection of biomarkers for diagnosis of diseases. The reversed-phase (RP) liquid chromatography (LC)/mass spectrometry (MS) is clearly the method of choice to analyze complex mixtures: the application of the method to proteomic analyses became the golden standard in the field. However, the presence of charged metabolites, especially, basic molecules containing amines in the metabolites makes it difficult to get good separation during the reversed-phase chromatography. In this study, we introduced a method using RPLC/MS. The dansyl chloride has been used in the N-terminal labeling of proteins and peptides, and its chemistry has been well studied. Dansylation of metabolites containing primary amine, secondary amine, or phenolic hydroxyl group, the presence of which deteriorate the chromatographic separation due to their interaction with the residual silanol group of the reversed-phase column material, not only improves the chromatographic separation but also increases the ionization efficiency during the electrospray ionization process. To increase the number of metabolite to be detected, the biological extracts were first separated using an ion-paring reversed-phase liquid chromatography with heptafluorobutyric acid as ion-paring reagent, and the individual fractions were subjected to the LC/MS analysis. This method was applied for acquiring metabolite profile from human urine samples. Analysis of the LC-MS data were performed by XCMS program (http://metlinscripps.edu/xcms), which automatically eliminated isotopic peaks, common adduct ions, multiply charged ions. Ion spectra differentially altered between sample groups were analyzed based on retention time and accurate mass matches. As the results, 416 ion spectra or metabolites were detected from nine ion-pair RPLC fractions. In addition, using a library of 220 amine- and phenol-containing metabolite standards, we were able to identify 58 metabolites containing amine and 28 amino acids. These studies can provide useful metabolomic information that has important implications for understanding the biology of disease state and identifying potential biomarkers.
|年月日||必須||2013年 5月 31日|