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Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides

Nature Protocols volume 14, pages 283–312 (2019)Cite this article

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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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Fig. 1: Procedure overview.
Fig. 2: Calibration curve preparation.
Fig. 3: Exemplary impairment of results by mycoplasma contamination.
Fig. 4: Chromatogram of different UV traces as represented in the QQQ quantitative analysis program.
Fig. 5: Chromatograms for m4dC and 15N2-m4dC.
Fig. 6: Chromatogram showing the analysis of hm5dC, ca5dC, m5dC, f5dC and 8oxodG with their corresponding isotopically labeled standards.
Fig. 7: Quantities of m5dC, hm5dC, f5dC, ca5dC and 8oxodG in small molecule–inducible neurogenin human-induced pluripotent stem cells lines (iNGNs) (8 d after induction of differentiation), untransfected HEK293T cells and HEK293T cells that were transfected for 24 h with a plasmid coding for Tet3.
Fig. 8: Exemplary calibration curve for m4dC.

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References

  1. Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).

    Article  CAS  Google Scholar 

  2. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  Google Scholar 

  3. Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. Int. Ed. Engl. 50, 7008–7012 (2011).

    Article  CAS  Google Scholar 

  4. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).

    Article  CAS  Google Scholar 

  5. He, Y.-F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303-1307 (2011).

  6. Wu, H. & Zhang, Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Gene. Dev. 25, 2436–2452 (2011).

    Article  CAS  Google Scholar 

  7. Branco, M. R., Ficz, G. & Reik, W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 13, 7–13 (2011).

    Article  Google Scholar 

  8. Langemeijer, S. M. C. et al. Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat. Genet. 41, 838–842 (2009).

    Article  CAS  Google Scholar 

  9. Ko, M. et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839–843 (2010).

    Article  CAS  Google Scholar 

  10. Jones, P. A. & Baylin, S. B. The epigenomics of cancer. Cell 128, 683-692 (2007).

  11. Pfaffeneder, T. et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat. Chem. Biol. 10, 574–581 (2014).

    Article  CAS  Google Scholar 

  12. Bachman, M. et al. 5-Formylcytosine can be a stable DNA modification in mammals. Nat. Chem. Biol. 11, 555–557 (2015).

    Article  CAS  Google Scholar 

  13. Iwan, K. et al. 5-Formylcytosine to cytosine conversion by C–C bond cleavage in vivo. Nat. Chem. Biol. 14, 72–78 (2018).

    Article  CAS  Google Scholar 

  14. Raiber, E.-A. et al. 5-Formylcytosine alters the structure of the DNA double helix. Nat. Struct. Mol. Biol. 22, 44–49 (2014).

    Article  Google Scholar 

  15. Song, C. X. et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153, 678–691 (2013).

    Article  CAS  Google Scholar 

  16. Kellinger, M. W. et al. 5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 19, 831–833 (2012).

    Article  CAS  Google Scholar 

  17. Zhu, C. et al. Single-cell 5-formylcytosine landscapes of mammalian early embryos and ESCs at single-base resolution. Cell Stem Cell 20, 720–731.e5 (2017).

    Article  CAS  Google Scholar 

  18. Hill, P. W. S., Amouroux, R. & Hajkova, P. DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: an emerging complex story. Genomics 104, 324–333 (2014).

    Article  CAS  Google Scholar 

  19. Yu, M. et al. Base-resolution detection of N(4)-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite sequencing. Nucleic Acids Res. 43, e148 (2015).

    Article  Google Scholar 

  20. Ehrlich, M. et al. DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine. Nucleic Acids Res. 13, 1399–1412 (1985).

    Article  CAS  Google Scholar 

  21. Arber, W. & Dussoix, D. Host specificity of DNA produced by Escherichia coli: I. Host controlled modification of bacteriophage λ. J. Mol. Biol. 5, 18–36 (1962).

    Article  CAS  Google Scholar 

  22. Wu, T. P. et al. DNA methylation on N6-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016).

    Article  CAS  Google Scholar 

  23. Huang, W. et al. Determination of DNA adenine methylation in genomes of mammals and plants by liquid chromatography/mass spectrometry. RSC Adv. 5, 64046–64054 (2015).

    Article  CAS  Google Scholar 

  24. Koziol, M. J. et al. Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nat. Struct. Mol. Biol. 23, 24–30 (2015).

    Article  Google Scholar 

  25. Liu, J. et al. Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig. Nat. Commun. 7, 13052 (2016).

    Article  CAS  Google Scholar 

  26. Schiffers, S. et al. Quantitative LC–MS provides no evidence for m6dA or m4dC in the genome of mouse embryonic stem cells and tissues. Angew. Chem. Int. Ed. Engl. 56, 11268–11271 (2017).

    Article  CAS  Google Scholar 

  27. Wang, D., Kreutzer, D. A. & Essigmann, J. M. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions. Mutat. Res. 400, 99–115 (1998).

    Article  CAS  Google Scholar 

  28. Cooke, M. S., Evans, M. D., Dizdaroglu, M. & Lunec, J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195–1214 (2003).

    Article  CAS  Google Scholar 

  29. Lunec, J. ESCODD: European standards committee on oxidative DNA damage. Free Radical Res. 29, 601–608 (1998).

    Article  CAS  Google Scholar 

  30. Murray, K. K. et al. Definitions of terms relating to mass spectrometry (IUPAC Recommendations 2013). Pure Appl. Chem. 85, 1515–1609 (2013).

    Article  CAS  Google Scholar 

  31. Oakeley, E. J. DNA methylation analysis: a review of current methodologies. Pharmacol. Ther. 84, 389–400 (1999).

    Article  CAS  Google Scholar 

  32. Taghizadeh, K. et al. Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nat. Protoc. 3, 1287–1298 (2008).

    Article  CAS  Google Scholar 

  33. Annesley, T. M. Ion suppression in mass spectrometry. Clin. Chem. 49, 1041–1044 (2003).

    Article  CAS  Google Scholar 

  34. Buhrman, D. L., Price, P. I. & Rudewiczcor, P. J . Quantitation of SR 27417 in human plasma using electrospray liquid chromatography–tandem mass spectrometry: a study of ion suppression.J. Am. Soc. Mass Spectrom 7, 1099–1105 (1996).

    Article  CAS  Google Scholar 

  35. Brückl, T., Globisch, D., Wagner, M., Müller, M. & Carell, T. Parallel isotope-based quantification of modified tRNA nucleosides. Angew. Chem. Int. Ed. Engl. 48, 7932–7934 (2009).

    Article  Google Scholar 

  36. Münzel, M. et al. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew. Chem. Int. Ed. Engl. 49, 5375–5377 (2010).

    Article  Google Scholar 

  37. Globisch, D. et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367 (2010).

    Article  CAS  Google Scholar 

  38. Pearson, D. et al. LC-MS based quantification of 2′-ribosylated nucleosides Ar(p) and Gr(p) in tRNA. Chem. Commun. 47, 5196–5198 (2011).

    Article  CAS  Google Scholar 

  39. Globisch, D. et al. Systems-based analysis of modified tRNA bases. Angew. Chem. Int. Ed. Engl. 50, 9739–9742 (2011).

    Article  CAS  Google Scholar 

  40. Wetzel, C. & Limbach, P. A. Global identification of transfer RNAs by liquid chromatography–mass spectrometry (LC–MS). J. Proteomics 75, 3450–3464 (2012).

    Article  CAS  Google Scholar 

  41. Schiesser, S. et al. Deamination, oxidation, and C–C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. J. Am. Chem. Soc. 135, 14593–14599 (2013).

    Article  CAS  Google Scholar 

  42. Schröder, A. S. et al. Synthesis of a DNA promoter segment containing all four epigenetic nucleosides: 5-methyl-, 5-hydroxymethyl-, 5-formyl-, and 5-carboxy-2′-deoxycytidine. Angew. Chem. Int. Ed. Engl. 53, 315–318 (2014).

    Article  Google Scholar 

  43. Thienpont, B. et al. Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Nature 537, 63–68 (2016).

    Article  CAS  Google Scholar 

  44. Rahimoff, R. et al. 5-Formyl- and 5-carboxydeoxycytidines do not cause accumulation of harmful repair intermediates in stem cells. J. Am. Chem. Soc. 139, 10359–10364 (2017).

    Article  CAS  Google Scholar 

  45. Turowski, M. et al. Deuterium isotope effects on hydrophobic interactions: the importance of dispersion interactions in the hydrophobic phase. J. Am. Chem. Soc. 125, 13836–13849 (2003).

    Article  CAS  Google Scholar 

  46. Kanao, E. et al. Isotope effects on hydrogen bonding and CH/CD−π interaction. J. Phys. Chem. 122, 15026–15032 (2018).

    CAS  Google Scholar 

  47. Liu, S. & Wang, Y. Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts. Chem. Soc. Rev. 44, 7829–7854 (2015).

    Article  CAS  Google Scholar 

  48. Lentini, A. et al. A reassessment of DNA-immunoprecipitation-based genomic profiling. Nat. Methods 15, 499–504 (2018).

    Article  CAS  Google Scholar 

  49. Shrivastava, A. & Gupta, V. B. HPLC: isocratic or gradient elution and assessment of linearity in analytical methods. J. Adv. Sci. Res. 3, 12–20 (2012).

    CAS  Google Scholar 

  50. Schellinger, A. P. & Carr, P. W. Isocratic and gradient elution chromatography: a comparison in terms of speed, retention reproducibility and quantitation. J. Chromatogr. A 1109, 253–266 (2006).

    Article  CAS  Google Scholar 

  51. Zhang, J. J. et al. Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry. Anal. Biochem. 413, 164–170 (2011).

    Article  CAS  Google Scholar 

  52. Contreras-Sanz, A. et al. Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC. Purinergic Signal. 8, 741–751 (2012).

    Article  CAS  Google Scholar 

  53. Yonekura, S., Iwasaki, M., Kai, M. & Ohkura, Y. High-performance liquid chromatographic determination of guanine and its nucleosides and nucleotides in biospecimens with precolumn fluorescence derivatization using phenylglyoxal. Anal. Sci. 10, 247–251 (1994).

    Article  CAS  Google Scholar 

  54. Giel-Pietraszuk, M. et al. Quantification of 5-methyl-2′-deoxycytidine in the DNA. Acta Biochim. Pol. 62, 281–286 (2015).

    Article  CAS  Google Scholar 

  55. Pitt, J. J. Principles and applications of liquid chromatography-mass spectrometry in clinical biochemistry. Clin. Biochem. Rev. 30, 19–34 (2009).

    PubMed  PubMed Central  Google Scholar 

  56. Niessen, W. M. A. State-of-the-art in liquid chromatography–mass spectrometry. J. Chromatogr. A 856, 179–197 (1999).

    Article  CAS  Google Scholar 

  57. Williamson, L. N. & Bartlett, M. G. Quantitative liquid chromatography/time-of-flight mass spectrometry. Biomed. Chromatogr. 21, 567–576 (2007).

    Article  CAS  Google Scholar 

  58. Payne, A. H. & Glish, G. L. Tandem mass spectrometry in quadrupole ion trap and ion cyclotron resonance mass spectrometers. Methods Enzymol. 402, 109–148 (2005).

    Article  CAS  Google Scholar 

  59. Tozuka, Z. et al. Strategy for structural elucidation of drugs and drug metabolites using (MS)n fragmentation in an electrospray ion trap. J. Mass Spectrom. 38, 793–808 (2003).

    Article  CAS  Google Scholar 

  60. Ens, W. & Standing, K. G. Hybrid quadrupole/time-of-flight mass spectrometers for analysis of biomolecules. Methods Enzymol. 402, 49–78 (2005).

    Article  CAS  Google Scholar 

  61. Commission Decision 2002/657/EC. Implementation of Council Directive 96/23/EC Concerning the Performance of Analytical Methods and the Interpretation of Results (Brussels, Official Journal of the European Communities, 2002).

  62. Zhao, Z. & Zhang, F. Sequence context analysis in the mouse genome: single nucleotide polymorphisms and CpG island sequences. Genomics 87, 68–74 (2006).

    Article  CAS  Google Scholar 

  63. Kellner, S. et al. Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability. Nat. Chem. Biol. 13, 888–894 (2017).

    Article  CAS  Google Scholar 

  64. Kellner, S. et al. Absolute and relative quantification of RNA modifications via biosynthetic isotopomers. Nucleic Acids Res. 42, e142 (2014).

    Article  Google Scholar 

  65. Amouroux, R. et al. De novo DNA methylation drives 5hmC accumulation in mouse zygotes. Nat. Cell Biol. 18, 225–233 (2016).

    Article  CAS  Google Scholar 

  66. Busskamp, V. et al. Rapid neurogenesis through transcriptional activation in human stem cells. Mol. Syst. Biol. 10, 760 (2014).

    Article  Google Scholar 

  67. Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992).

    Article  CAS  Google Scholar 

  68. Collins, A. R., Cadet, J., Mőller, L., Poulsen, H. E. & Viña, J. Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch. Biochem. Biophys. 423, 57–65 (2004).

    Article  CAS  Google Scholar 

  69. ESCODD. Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus. Carcinogenesis 23, 2129–2133 (2002).

    Article  Google Scholar 

  70. Ravanat, J.-L. et al. Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up. Carcinogenesis 23, 1911–1918 (2002).

    Article  CAS  Google Scholar 

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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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  1. These authors contributed equally: Franziska R. Traube, Sarah Schiffers, Katharina Iwan.

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  1. Center for Integrated Protein Science Munich (CiPSM), Department of Chemistry, Ludwig-Maximilians–Universität München, Munich, Germany

    Franziska R. Traube, Sarah Schiffers, Katharina Iwan, Stefanie Kellner, Fabio Spada, Markus Müller & Thomas Carell

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Contributions

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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Correspondence to Thomas Carell.

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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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