Determination of Site Selectivity of Different Carcinogens for Preferential Mutational Hot Spots in Oligonucleotide Fragments by Ion-Pair Reversed-Phase Nano Liquid Chromatography Tandem Mass Spectrometry. Issue 1 (February 2014) - Sharma, Vaneet K.
Ion-pair reversed-phase nano liquid chromatography coupled with nanospray ion trap mass spectrometry was used to investigate site selectivity of the known carcinogens N -acetoxy-2-acetylaminofluorene, N -hydroxy-4-aminobiphenyl and (±)- anti -benzo[ a ]pyrene diol epoxide with the synthetic double-strand 14-mer long oligonucleotide fragment of the p53 gene containing two mutational hot-spot codons (5′- P- ACC 155 CGC 156 GTC 157 CGC 158 GC/5′- GCG CGG ACG CGG GT) . The investigation was performed using a monolithic polystyrene divinylbenzene capillary column and triethylammonium bicarbonate as an ion-pair reagent. The exact location of the carcinogen on the modified oligonucleotide backbone was determined using characteristic collision-induced dissociation fragmentation patterns obtained under negative-ion mode ionization. In all these cases, the adducted, isomeric oligonucleotides formed were chromatographically resolved and structural identification was performed without any prior deoxyribonucleic acid cleavage or hydrolysis. The knowledge of the site specificity of a carcinogen, especially at purported mutational hot spots, is of paramount importance (1) in establishing the identity of biomarkers for an early risk assessment of the formed DNA adducts, (2) developing repair mechanisms for the formed carcinogen adducted DNA, and (3) understanding the nature of the covalent bond formed and mapping the frequency of the adduction process.