分析および生物分析技術ジャーナル

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A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases

Feng Jin, Salil Kumar Bhowmik, Vasanta Putluri, Franklin Gu, Jie Gohlke, Friedrich Carl Von Rundstedt, Subhamoy Dasgupta, Rashmi Krishnapuram, Bert W. O’Malley, Arun Sreekumar and Nagireddy Putluri

The growth of cancer cells relies more on increased proliferation and autonomy compared to non-malignant cells. The rate of de novo nucleotide biosynthesis correlates with cell proliferation rates. In part, glutamine is needed to sustain high rates of cellular proliferation as a key nitrogen donor in purine and pyrimidine nucleotide biosynthesis. In addition, glutamine serves as an essential substrate for key enzymes involved in the de novo synthesis of purine and pyrimidine nucleotides. Here, we developed a novel liquid chromatography (LC-MS) to quantify glutaminederived [15N] nitrogen flux into nucleosides and nucleobases (purines and pyrimidines). For this, DNA from 5637 bladder cancer cell line cultured in 15N labelled glutamine and then enzymatically hydrolyzed by sequential digestion. Subsequently, DNA hydrolysates were separated by LC-MS and Selected Reaction Monitoring (SRM) was employed to identify the nucleobases and nucleosides. Thus, high sensitivity and reproducibility of the method make it a valuable tool to identify the nitrogen flux primarily derived from glutamine and can be further adaptable for high throughput analysis of large set of DNA in a clinical setting.