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Method for the rapid detection and molecular characterization of DNA alkylating agents by MALDI-TOF mass spectrometry
Metabolic activation of polycyclic aromatic hydrocarbons (PAH) may cause DNA adduct formation. While these are commonly detected by the P-32-postlabeling assay, this method is not informative on the chemical nature of the alkylating agent. Here we report a simple and reliable method that employs MALDI-TOF-MS with 2,5-dihydroxybenzoic acid (DHB) matrix layer (ML) sample preparations for the detection and structural characterization of PAH-DNA adducts. The method involves the enzymatic digestion of DNA to 2'-deoxynucleotides followed by solid phase extraction to remove salt and other contaminants prior to MALDI-MS analysis. By collision induced dissociation (CID) structurally relevant fragments are obtained to permit characterization of the alkylating molecules and the adducted nucleotide. Next to guanosine, adenosine and cytidine adducts formed from reactions with (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) are identified at a sensitivity of <100 fmol and a massaccuracy of <10 ppm. Studies with (+/-)-anti-benzo[c]chrysene-9,10-diol-11,12-epoxide (B[c]ChDE) further document the versatility and usefulness of the method. When compared with the P-32-postlabeling assay MALDI-MS only indentified deoxycytidine as well nucleoside and dinucleotides adducts. Therefore, this sensitive method enables molecular specification and characterization of adducted nucleotides and of the alkylating agent, and thus, provides comprehensive information that is beyond the P-32-postlabeling assay.