Journal of Analytical & Bioanalytical Techniques
Open Access

Research Article

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

Feng Jin^1,4#, Salil Kumar Bhowmik^2,4#, Vasanta Putluri^1,4, Franklin Gu³, Jie Gohlke^2,4, Friedrich Carl Von Rundstedt^5,6, Subhamoy Dasgupta², Rashmi Krishnapuram², Bert W. O’Malley^2,4, Arun Sreekumar^1,2,3,4 and Nagireddy Putluri^1,2,4*

¹Advanced Technology Core, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA

²Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA

³Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA

⁴Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA

⁵Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA

⁶Helios Klinikum, Witten-Herdecke University, Wuppertal, Germany

^#Authors are contributed equally in this work

*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
Tel: 713-798-3139
E-mail: putluri@bcm.edu

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.

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