Accumulating evidence suggests that DNA lesion-induced
DNA-
protein cross-links (DPCs) interrupt normal
DNA metabolic processes, such as transcription, replication, and repair, resulting in profound biological consequences, including the development of many human diseases, such as
cancers. Although apurinic/apyrimidinic (AP) sites are among the most predominant DNA lesions and are in close proximity to the
histone proteins that they wrap around in the
nucleosome, knowledge of the chemical structure or biological consequences of their associated DPCs is limited in part due to a lack of sensitive and selective analytical methods. We developed liquid chromatography-tandem mass spectrometry coupled with a stable
isotope dilution method for rigorous quantitation of DPCs formed by reacting
a DNA AP site with a
lysine residue. In combination with chemical derivatization with fluorenylmethoxycarbonyl
chloride to form a hydrophobic conjugate, the developed LC-MS/MS method allows sensitive detection of AP site-Lys cross-links down to sub-1 adduct per 106 nt. After validation using a synthetic AP site-
lysine-cross-linked
peptide and an
oligodeoxyribonucleotide, the method was used to determine the concentration of AP site-
lysine cross-links in hot
acid-treated
DNA and in human cells exposed to
methyl methanesulfonate.