Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry.
High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.
Regulus
Peggy
P
Laboratoire Lésions des Acides Nucléiques, DRFMC/SCIB CEA Grenoble, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France.
Spessotto
Sébastien
S
Gateau
Mathilde
M
Cadet
Jean
J
Favier
Alain
A
Ravanat
Jean-Luc
JL
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000528:radiation effects*; D004249:DNA Damage; D004252:DNA Mutational Analysis / Q000379:methods*; D005720:Gamma Rays; D006801:Humans; D008970:Molecular Weight; D009000:Monocytes / Q000528:radiation effects*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004247:DNA; C427203:calf thymus DNA