An LC-ESI-MS/MS technique for quantifying polar oxidized guanine nucleotides from DNA digests

TOXI 48

Peter G. Slade, slade@mit.edu1, John S. Wishnok, wishnok@mit.edu1, and Steven R. Tannenbaum2. (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Ave, 56-731, Cambridge, MA 02139, (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 56-731A, Cambridge, MA 02139
8-oxo-2'-deoxyguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at guanine. Unfortunately, 8-oxoG has been an inconsistent marker of cellular DNA oxidation, since it has a lower reduction potential than guanine and is susceptible to further or hyper oxidation. The hyper-oxidation products of guanine in DNA include spiroiminodihydantoin (Sp) and guanidinohyantoin (Gh), which are extremely polar, poorly ionized by ESI, and likely form in cellular DNA at a ratio of less than 1 in 10^6 guanines. They are consequently resistant to analysis, and have yet to be identified in a mammalian cellular system. A technique has been developed using an Amide-80 column and capillary HPLC to separate phosphorylated nucleotides. Hyper-oxidized guanine lesions have been identified/quantified by ion trap ESI-MS/MS (MRM), and we are now assessing whether this technique has the sensitivity and specificity to identify Sp and Gh in cellular DNA.
 

Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster

Division of Chemical Toxicology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007