Feng Jin (Presenter)
Baylor College of Medicine
Bio: This is Feng Jin. I went to Ohio University to study portable mass spectromter with Dr. Glen Jackson at Ohio University. Then I moved to West Virginia University with Dr. Glen Jackson. After I graduated with my Ph.D in analytical chemistry, I join Dr. Nagireddy Putluri lab to study Metabolomics with LC-MS.
Authorship: Jin, Feng$1,4, Bhowmik, Salil K. $2,4, Putluri, Vasanta 1,4, Gu, Franklin 3, Gohlke, Jie 2,4, Rundstedt, Friedrich C. 5,6, Dasgupta, Subhamoy 2, Krishnapuram, Rashmi 2, O’Malley, Bert W. 2,4, Sreekuma
1Advanced Technology Core, 2Department of Molecular and Cell Biology and Dan L. Duncan Cancer Center, 3Department of Biochemistry, 4Alkek Center for Molecular Discovery, 5Scott Department of Urology,
| Short Abstract A novel liquid chromatography mass spectrometry (LC-MS) method is developed to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases. This method will be a valuable tool to identify the nitrogen flux derived from glutamine and it can be further adaptable for high throughput analysis of large set of DNA in a clinical setting. |
Long Abstract
Introduction:
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 mass spectrometry (LC-MS) method to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases (purines and pyrimidines).
Methods:
We isolated 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.
Results:
We observe the CID spectrum of [M+H]+ (m/z 136) of adenine (A) (purine) shows product ions corresponding to the [M+H-2NCH=NH]+ ion (m/z 92) and that of [M+H]+ of guanine (G) (purine)) shows [M+H-NH3]+ ion (m/z 8 135). The CID mass spectrum of [M+H]+ of thymine (T) (pyrimidine). The fragment ions correspond to the [M+H-NH3]+ ion (m/z 110). The CID mass spectrum of [M+H]+ of cytosine (C) (pyrimidine) shows fragment ions corresponding to the [M+H-NHCO]+ ion (m/z 69). The CID mass spectrum of [M+H]+ of uracil (U) (pyrimidine) shows fragment ions corresponding to the [M+H-NHCO]+ ion (m/z 70). As expected, the ESI source, with the mass spectrometer in positive ion mode, gave protonated molecules and also fragment ions. We chose to monitor the fragment ions of the compounds of interest rather than the protonated molecules and further SRM approach to quantify the levels of nucleobases and nucleosides. Also, [15N] Glutamine flux for 12h, 24h, 48h, and 72h was determined. Collectively, results above clearly suggest the glutamine nitrogen feed in to the nucleosides.
Conclusion:
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.
References & Acknowledgements:
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| Salary | no | |
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IP Royalty: no
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