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 ³²P-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 mass accuracy 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 ³²P-postlabeling assay MALDIMS 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 ³²P-postlabeling assay.
Garaguso
Ignazio
I
Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
Halter
Roman
R
Krzeminski
Jacek
J
Amin
Shantu
S
Borlak
Jürgen
J
D000477:Alkylating Agents / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004247:DNA / Q000032:analysis*; D018736:DNA Adducts / Q000032:analysis*; D006868:Hydrolysis; D015394:Molecular Structure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D000477:Alkylating Agents; D018736:DNA Adducts; D004247:DNA; C427203:calf thymus DNA