DNA-Protein Cross-Links Formed by Reacting Lysine with Apurinic/Apyrimidinic Sites in DNA and Human Cells: Quantitative Analysis by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope Dilution

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 10(6) 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.