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
Recommended Citation
Li, Jie, Elvis M. K. Leung, Martin M. F. Choi, and Wan Chan. "Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA." Analytical and Bioanalytical Chemistry 405.12 (2013): 4059-4066.