Abstract
DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme–based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.
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Acknowledgements
We thank the Deutsche Forschungsgemeinschaft for funding through SFB749, SFB1032, SPP1784 and CA275-11/1. Further support is acknowledged from the Excellence Cluster CiPSM (Center for Integrated Protein Science). F.R.T. thanks the Boehringer Ingelheim Fonds for her PhD fellowship. S.K. thanks the Fonds der Chemischen Industrie for the Liebig Fellowship. We thank T. Pfaffeneder for early method development and helpful input. We thank S. Michalakis (Department of Pharmacy, Ludwig-Maximilians–Universität München) for providing mouse tissues.
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F.R.T., S.S. and K.I. designed and developed the protocol. S.K. provided expertise on mass spectrometry. F.S. developed the DNA isolation protocol. M.M. designed graphics. T.C. supervised and designed the studies. All authors participated in writing the manuscript.
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Key references using this protocol
Pfaffeneder, T. et al. Nat. Chem. Biol. 10, 574–581 (2014): https://www.nature.com/articles/nchembio.1532
Iwan, K. et al. Nat. Chem. Biol. 14, 72–78 (2018): https://www.nature.com/articles/nchembio.2531
Schiffers, S. et al. Angew. Chem. Int. Ed. Engl. 56, 11268–11271 (2017): https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201700424
Rahimoff, R. et al. J. Am. Chem. Soc. 139, 10359–10364 (2017): https://pubs.acs.org/doi/10.1021/jacs.7b04131
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Traube, F.R., Schiffers, S., Iwan, K. et al. Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides. Nat Protoc 14, 283–312 (2019). https://doi.org/10.1038/s41596-018-0094-6
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DOI: https://doi.org/10.1038/s41596-018-0094-6
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