Quantification of a Novel DNA-Protein Cross-Link Product Formed by Reacting Apurinic/Apyrimidinic Sites in DNA with Cysteine Residues in Protein by Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

Emerging evidence suggests that cross-links formed by reacting DNA lesions with proteins may play a significant role in the pathophysiology of human cancer and degenerative diseases. The goal of this study was to develop a method involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with the stable isotope-dilution method to quantify DNA-protein cross-link (DPC). A novel type of cross-link involving a S-glycosidic linkage formed by reacting an abasic site in DNA with the cysteine residues in protein was targeted in this study. The method entails hydrolysis of the cross-link to a 2'-deoxyribose-cysteine adduct, addition of isotopically labeled internal standard, and quantitation by LC-MS/MS analysis. The accuracy and precision of the method were evaluated with a synthetic peptide containing the cross-link. The validated method was then applied to quantitate the levels of the DNA-protein cross-link in vitro and in HeLa cells exposed to alkylating agent methylmethanesulfonate (MMS). The analysis detected dosage-dependent formation of the cross-link in both purified DNA (6.0 ± 0.6 DPC per 10 nt μM MMS) and in human cells (7.8 ± 1.2 DPC per 10 nt mM MMS). With the abasic site being one of the most common DNA lesions produced continuously by multiple pathways, the results provide significant new knowledge for better understanding the potential biological implications of its associated DNA-protein cross-link.