title: Optimized enzymatic hydrolysis of DNA for LC-MS/MS analyses of adducts of 1-methoxy-3-indolylmethyl glucosinolate and methyleugenol.
abstract: Mass spectrometric analyses of DNA adducts usually require enzymatic digestion of the DNA to nucleosides. The digestive enzymes used in our laboratory included a calf spleen phosphodiesterase, whose marketing was stopped recently. Using DNA adducted with bioactivated methyleugenol and 1-methoxy-3-indolylmethyl glucosinolate-each forming dA and dG adducts-we demonstrate that replacement of calf spleen phosphodiesterase (Merck) with bovine spleen phosphodiesterase (Sigma-Aldrich) leads to unchanged results. Enzyme levels used for DNA digestion are extremely variable in different studies. Therefore, we sequentially varied the level of each of the three enzymes used. All dose (enzyme)-response (adduct level) curves involved a long plateau starting below the enzyme levels employed previously. Thus, we could reduce the amounts of micrococcal nuclease, phosphodiesterase, and alkaline phosphatase for quantitative DNA digestion by factors of 4, 2, and 333, respectively, compared to our previous protocols. Moreover, we observed significant phosphatase activity of both phosphodiesterase preparations used, which may affect the recovery of adducts with methods requiring digestion to 2'-deoxynucleoside-3'-monophosphates (e.g., (32)P-postlabeling). In addition, the phosphodiesterase from Sigma-Aldrich, but not that from Merck, deaminated dA. This was irrelevant for the dA adducts studied, involving bonding at N(6), but might complicate the analysis of other dA adducts.
authors.affiliation: German Institute of Human Nutrition, Potsdam-Rehbrücke, 14558 Nuthetal, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Schumacher
authors.forename: Kristin
authors.initials: K
authors.lastname: Herrmann
authors.forename: Simone
authors.initials: S
authors.lastname: Florian
authors.forename: Wolfram
authors.initials: W
authors.lastname: Engst
authors.forename: Hansruedi
authors.initials: H
authors.lastname: Glatt
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D005054:Eugenol / Q000031:analogs & derivatives* / Q000737:chemistry; D005961:Glucosinolates / Q000737:chemistry*; D006868:Hydrolysis; D007211:Indoles / Q000737:chemistry*; D008836:Micrococcal Nuclease / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D018736:DNA Adducts; D003839:Deoxyadenosines; D005961:Glucosinolates; D007211:Indoles; D010761:Phosphorus Radioisotopes; C005223:methyleugenol; D005054:Eugenol; C036025:4-methoxy-3-indolylmethyl glucosinolate; D004247:DNA; D000469:Alkaline Phosphatase; D008836:Micrococcal Nuclease; D010727:Phosphoric Diester Hydrolases