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Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry

Nature Protocols volume 3, pages 1287–1298 (2008)Cite this article

Abstract

The analysis of damage products as biomarkers of inflammation has been hampered by a poor understanding of the chemical biology of inflammation, the lack of sensitive analytical methods and a focus on single chemicals as surrogates for inflammation. To overcome these problems, we developed a general and sensitive liquid chromatographic tandem mass spectrometry (LC/MS-MS) method to quantify, in a single DNA sample, the nucleoside forms of seven DNA lesions reflecting the range of chemistries associated with inflammation: 2′-deoxyuridine, 2′-deoxyxanthosine and 2′-deoxyinosine from nitrosative deamination; 8-oxo-2′-deoxyguanosine from oxidation; and 1,N2-etheno-2′-deoxyguanosine, 1,N6-etheno-2′-deoxyadenosine and 3,N4-etheno-2′-deoxycytidine arising from reaction of DNA with lipid peroxidation products. Using DNA purified from cells or tissues under conditions that minimize artifacts, individual nucleosides are purified by HPLC and quantified by isotope-dilution, electrospray ionization LC/MS-MS. The method can be applied to other DNA damage products and requires 4–6 d to complete depending upon the number of samples.

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Figure 1: The chemical biology of inflammation.
Figure 2: DNA damage products associated with the chemical mediators of inflammation.
Figure 3
Figure 4: Reversed-phase HPLC separation of normal and modified nucleoside standards.

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Acknowledgements

We gratefully acknowledge financial support for this work from the National Institutes of Health (ES002109, CA116318, CA26735, ES59790, CA103146, CA110261, S10RR023783).

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Author notes

  1. Bo Pang & Min Dong

    Present address: Present addresses: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142, USA (B.P.) and Novartis Pharma AG, Auhafenstrasse, Muttenz, Auhafenstrasse CH-4132 Muttenz, Switzerland (M.D.).,

Authors and Affiliations

  1. Center for Environmental Health Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Koli Taghizadeh, Elaine Plummer & Peter C Dedon

  2. Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Jose L McFaline, Bo Pang, Matthew Sullivan, Min Dong & Peter C Dedon

Authors
  1. Koli Taghizadeh
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Correspondence to Peter C Dedon.

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Taghizadeh, K., McFaline, J., Pang, B. 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). https://doi.org/10.1038/nprot.2008.119

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