Abstract
Physiological and chemically induced modifications to nucleosides are common in both DNA and RNA. Physiological forms of these modifications play critical roles in gene expression, yet aberrant marks, if left unrepaired, may be associated with increased genome instability. Due to the low prevalence of these marks in most samples of interest, a highly sensitive method is needed for their detection and quantitation. High-performance liquid chromatography, coupled to mass spectrometry (HPLC-MS), provides this high degree of sensitivity while also being adaptable to nearly any modified nucleoside of interest and still maintaining exquisite specificity. In this chapter, we demonstrate how to use HPLC-MS to analyze the catalytic activity of a nucleic acid demethylase, to quantify the prevalence of N6-methyladenosine from RNA, and to determine the kinetics of alkylation damage repair.
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Acknowledgments
Work in our laboratory is supported the by the NIH (R01 CA193318, R01 CA227001, and P01 CA092584), the American Cancer Society research scholar program (RSG-18-156-01-DMC), and the Alvin J. Siteman Cancer Center Investment Program, which is supported by the Foundation for Barnes-Jewish Hospital Cancer Frontier Fund and the National Cancer Institute, Cancer Support Grant P30 CA091842.
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Rodell, R., Tsao, N., Ganguly, A., Mosammaparast, N. (2022). Use of High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) to Quantify Modified Nucleosides. In: Mosammaparast, N. (eds) DNA Damage Responses. Methods in Molecular Biology, vol 2444. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2063-2_8
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DOI: https://doi.org/10.1007/978-1-0716-2063-2_8
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