Abstract
Reliable quantitative analysis of DNA modification using liquid chromatography coupled with tandem mass spectrometry requires stable isotope-labeled internal standards. Only some of them are commercially available. Here we present a method allowing for the synthesis of [13C10,15N2]-5-methyl-2′-deoxycytidine from [13C10,15N2]-2′-deoxythymidine. We also describe an approach for the oxidation of [13C10,15N2]-5-methyl-2′-deoxycytidine and [13C10,15N2]-2′-deoxythymidine with Na2S2O8, leading to the generation of [13C10,15N2]-5-formyl-2′-deoxycytidine, [13C10,15N2]-5-carboxy-2′-deoxycytidine or [13C10,15N2]-5-(hydroxymethyl)-2′-deoxyuridine, correspondingly. Moreover, we provide optimized protocols for the oxidation of [13C5,15N2]-thymine to [13C10,15N2]-5-hydroxymethyluracil, [13C10,15N2]-5-formyluracil, and [13C10,15N2]-5-carboxyuracil using Na2S2O8.
Marta Starczak and Aleksandra Skalska contributed equallly to this work.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Vogl J, Pritzkow W (2010) Isotope dilution mass spectrometry - a primary method of measurement and its role for RM certification. Mapan J Metrol Soc I 25(3):135–164. https://doi.org/10.1007/s12647-010-0017-7
Meija J, Mester Z (2008) Paradigms in isotope dilution mass spectrometry for elemental speciation analysis. Anal Chim Acta 607(2):115–125. https://doi.org/10.1016/j.aca.2007.11.050
Dizdaroglu M (1993) Quantitative determination of oxidative base damage in DNA by stable isotope-dilution mass spectrometry. FEBS Lett 315(1):1–6. https://doi.org/10.1016/0014-5793(93)81120-o
Jennings ME 2nd, Matthews DE (2005) Determination of complex isotopomer patterns in isotopically labeled compounds by mass spectrometry. Anal Chem 77(19):6435–6444. https://doi.org/10.1021/ac0509354
Borland K, Diesend J, Ito-Kureha T et al (2019) Production and application of stable isotope-labeled internal standards for RNA modification analysis. Genes (Basel) 10(1). https://doi.org/10.3390/genes10010026
Gackowski D, Starczak M, Zarakowska E et al (2016) Accurate, direct, and high-throughput analyses of a broad spectrum of endogenously generated DNA base modifications with isotope-dilution two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry: possible clinical implication. Anal Chem 88(24):12128–12136. https://doi.org/10.1021/acs.analchem.6b02900
Raiber EA, Hardisty R, van Delft P et al (2017) Mapping and elucidating the function of modified bases in DNA (vol 1, 0069, 2017). Nat Rev Chem 1(11)
Divakar KJ, Reese CB (1982) 4-(1,2,4-Triazol-1-yl)- and 4-(3-nitro-1,2,4-triazol-1-yl)-1-(β-D-2,3,5-tri-O-acetylarabinofuranosyl)pyrimidin-2(1H)-ones. Valuable intermediates in the synthesis of derivatives of 1-(β-D-arabinofuranosyl)cytosine (ara-C). J Chem Soc 1:1171–1176. https://doi.org/10.1039/p19820001171
Burdzy A, Noyes KT, Valinluck V et al (2002) Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA. Nucleic Acids Res 30(18):4068–4074. https://doi.org/10.1093/nar/gkf520
Rahman AAHA, Wada T, Saigo K (2001) Facile methods for the synthesis of 5-formylcytidine. Tetrahedron Lett 42(6):1061–1063
Patel JP, Sowers ML, Herring JL et al (2015) Measurement of postreplicative DNA metabolism and damage in the rodent brain. Chem Res Toxicol 28(12):2352–2363. https://doi.org/10.1021/acs.chemrestox.5b00359
Mantele W, Deniz E (2017) UV-VIS absorption spectroscopy: Lambert-Beer reloaded. Spectrochim Acta A Mol Biomol Spectrosc 173:965–968. https://doi.org/10.1016/j.saa.2016.09.037
Schmid FX (2001) Biological macromolecules: UV-visible spectrophotometry. In: eLS. https://doi.org/10.1038/npg.els.0003142
Acknowledgments
This work was supported by the National Science Centre Grants No.: 2015/17/B/NZ5/00640, 2015/19/B/NZ5/02208 and 2017/27/B/NZ7/01487. The Authors are a members of University Center of Excellence “Towards Personalized Medicine” operating under Excellence Initiative – Research University.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Starczak, M., Skalska, A., Rozalski, R., Olinski, R., Gackowski, D. (2021). Preparation of Internal Standards for 2D-UPLC-MS/MS Quantification of Noncanonical DNA Bases. In: Ruzov, A., Gering, M. (eds) DNA Modifications. Methods in Molecular Biology, vol 2198. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0876-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0876-0_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0875-3
Online ISBN: 978-1-0716-0876-0
eBook Packages: Springer Protocols