Document Type

Journal Article

Department/Unit

Department of Chemistry

Title

Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA

Language

English

Abstract

Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography- coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/109 nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA. © Springer-Verlag Berlin Heidelberg 2012.

Keywords

Apurinic/apyrimidinic sites, Isotopedilution . Pentafluorophenylhydrazine .Derivatization, LC-MS/MS

Publication Date

2013

Source Publication Title

Analytical and Bioanalytical Chemistry

Volume

405

Issue

12

Start Page

4059

End Page

4066

Publisher

Springer Verlag

DOI

10.1007/s00216-013-6823-3

Link to Publisher's Edition

http://dx.doi.org/10.1007/s00216-013-6823-3

ISSN (print)

16182642

ISSN (electronic)

16182650

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