title: A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases.
abstract: 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 glutamine-derived [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.
authors.affiliation: Advanced Technology Core, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Jin
authors.affiliation: Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Salil Kumar
authors.initials: SK
authors.lastname: Bhowmik
authors.affiliation: Advanced Technology Core, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Vasanta
authors.initials: V
authors.lastname: Putluri
authors.affiliation: Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Franklin
authors.initials: F
authors.lastname: Gu
authors.affiliation: Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Gohlke
authors.affiliation: Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA; Helios Klinikum, Witten-Herdecke University, Wuppertal, Germany.
authors.forename: Friedrich Carl
authors.initials: FC
authors.lastname: Von Rundstedt
authors.affiliation: Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Subhamoy
authors.initials: S
authors.lastname: Dasgupta
authors.affiliation: Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Krishnapuram
authors.affiliation: Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Bert W
authors.initials: BW
authors.lastname: O'Malley
authors.affiliation: Advanced Technology Core, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Arun
authors.initials: A
authors.lastname: Sreekumar
authors.affiliation: Advanced Technology Core, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA; Alkek Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA.
authors.forename: Nagireddy
authors.initials: N
authors.lastname: Putluri
keywords: Bladder cancer cell lines; Glutamine nitrogen flux; Nucleosides and nucleobases biosynthesis; Tandem mass spectrometry