Purification and recovery of bulky hydrophobic DNA adducts.
For many years (32)P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of mass spectrometry in mind, the current research presents a new method to quantitatively purify bulky hydrophobic DNA adducts at levels that are pertinent to ongoing DNA adduct research in human health and environmental fields. This method was demonstrated with benzo[a]pyrene adducts. Purification was accomplished with the use of small columns (7.5-mm frits) with an 11 mg bed of polystyrene-divinlybenzene beads which retained the adducts while permitting the nonadducted nucleotides to be washed out with water. Subsequently, the adducts were eluted with 50% MeOH and the sample was reduced in volume in an evacuated centrifuge. Purification was demonstrated at adduct levels ranging from 4 adducts in 10(6) nonadducted nucleotides to 4 in 10(8). For these levels, analyses by capillary electrophoresis with sample stacking and UV detection determined that recoveries ranged from 91 to 54%, respectively. The adduct quantities isolated should be sufficient to allow the use of current MS capabilities that are linked on-line to separation methodologies such as capillary electrophoresis, capillary electrochromatography, and high-pressure liquid chromatography.
U. S. EPA AED, 27 Tarzwell Drive, Narragansett, Rhode Island 02882, USA.
C B
CB
Norwood
D000818:Animals; D001564:Benzo(a)pyrene / Q000302:isolation & purification; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D018736:DNA Adducts / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary; D006801:Humans; D013058:Mass Spectrometry; D000432:Methanol; D011137:Polystyrenes; D013056:Spectrophotometry, Ultraviolet
D018736:DNA Adducts; D011137:Polystyrenes; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; C003771:Amberlite XAD-2 resin; D000432:Methanol