A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases
Feng Jin1,4#, Salil Kumar Bhowmik2,4#, Vasanta Putluri1,4, Franklin Gu3, Jie Gohlke2,4, Friedrich Carl Von Rundstedt5,6, Subhamoy Dasgupta2, Rashmi Krishnapuram2, Bert W. O’Malley2,4, Arun Sreekumar1,2,3,4 and Nagireddy Putluri1,2,4*
- *Corresponding Author:
- Nagireddy Putluri
Assistant Professor, Director of Metabolomics
Department of Molecular and Cell Biology
Alkek Center for Molecular Discovery
Baylor College of Medicine
One Baylor Plaza Houston, TX-77030, USA
E-mail: [email protected]
Received date: August 19, 2015; Accepted date: August 26, 2015; Published date: August 30, 2015
Citation: Jin F, SK Bhowmik, Putluri V, Gu F, Gohlke J, et al. (2015) A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases. J Anal Bioanal Tech 6:267 doi:10.4172/2155-9872.1000267
Copyright: © 2015 Jin F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.