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Measurement of methylated metabolites using liquid chromatography-mass spectrometry and its biological application

Ambati, Chandrashekar R., Vantaku, Venkatrao, Donepudi, Sri Ramya, Amara, Chandra Sekhar, Ravi, Shiva Shankar, Mandalapu, Akhil, Perla, Maharajni, Putluri, Vasanta, Sreekumar, Arun, Putluri, Nagireddy
Analytical methods 2018 v.11 no.1 pp. 49-57
amino acids, cell lines, high performance liquid chromatography, hydrophilic interaction chromatography, hydrophobic bonding, ionization, mass spectrometry, metabolic diseases, metabolites, metabolomics, methylation, monitoring, neoplasm cells, nucleotides, organic acids and salts, standard deviation, urinary bladder neoplasms
Methylation aberrations play an important role in many metabolic disorders including cancer. Methylated metabolites are direct indicators of metabolic aberrations, and currently, there is no liquid chromatography-mass spectrometry (LC-MS) based method available to cover all classes of methylated metabolites at low detection limits. In this study, we have developed a method for the detection of methylated metabolites, and its biological application. In this approach, we used a HILIC based HPLC method combined with MS to measure methylated organic acids, amino acids, and nucleotides. These metabolites were separated from each other by their hydrophobic interactions and analyzed using a targeted metabolomics approach by single reaction monitoring in positive and negative modes of electrospray ionization. These metabolites were quantified, and the interday reproducibility was <10% relative standard deviation. Furthermore, by applying this method, we identified high levels of methylated metabolites in bladder cancer cell lines compared to benign cells. In vitro treatment of cancer cells with a methylation inhibitor, 5-aza-2′-deoxycytidine, showed a decrease in these methylated metabolites. These data indicate that HPLC analysis using this HILIC based method could be a powerful tool for measuring methylated metabolites in biological specimens. This method is rapid, sensitive, selective, and precise to measure methylated metabolites.