Protocol abstract
Nature Protocols 3, - 1287 - 1298 (2008)
Published online: 17 July 2008 | doi:10.1038/nprot.2008.119
Subject Categories: Genetic analysis | Spectroscopy and structural analysis
Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry
Koli Taghizadeh1, Jose L McFaline2, Bo Pang2,3, Matthew Sullivan2, Min Dong2,3, Elaine Plummer1 & Peter C Dedon1,2
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.
- Center for Environmental Health Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
- Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
- 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.).
Correspondence to: Peter C Dedon1,2 e-mail: pcdedon@mit.edu
nature-products
A-Z product listing
- 1,N2-Etheno-2'-deoxyguanosine(AREL Biosynth. Inc.)
- 1,N6-Etheno-2'-deoxyadenosine(Sigma Chemical Co.)
- 15-ml Falcon tubes(VWR)
- 2'-Deoxyadenosine(Sigma Chemical Co.)
- 2'-Deoxycytidine(Sigma Chemical Co.)
- 2'-Deoxyguanosine(Sigma Chemical Co.)
