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Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA−DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA−protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5′-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on …