| This dissertation describes an analytical approach for the sequencing oligodeoxynucleotides (ODN) modified by arylamine groups. Both 3'- and 5'-exonucleases were utilized to generate the mass ladders for sequencing ODNs. The digestion conditions were optimized to process at the speed compatible with HPLC analysis time. This is the first application of HPLC-MS and exonuclease digestion for sequencing ODNs. This is also the first work that has successfully identified the sequence of DNA carcinogen adducts that have three or four reactive sites, as well as multiple carcinogen modifications, using mass spectrometry (MS).; Enzymatic digestion was carried out with submicrogram quantities of ODNs in the autosampler used for the HPLC-MS analyses. An HPLC on-line purification step was used to remove salt adducts before ESI-MS analysis. HPLC analysis were carried out at a 200 muL/min flow rate using a microbore short C18 column (1x50mm). A novel, ion-pairing reagent, dimethylbutylamine (DMBA), was used to enhance the separation and retention of ODNs. The mobile phase is slightly acidic, which minimizes the number of charge states associated with a potential ODN ion. Acetonitrile with mixed with the eluent post column to enhance the ESI efficiency. This new HPLC-MS method provides a 10 3 enhancement in sensitivity (10 femtomole) than methods previously reported in the literature. The enhanced sensitivity permits sequencing a nona ODN with only a few micrograms of sample (< 2 nmol). Using this method, we have identified the sequences of more than 90 nona ODNs containing one to four 2'-deoxyguanosines with one or two 2-aminofluorene (7-fluoro-2-aminofluorene) modifications.; A new strategy to deconvolute multiply charged peaks observed in the ESI mass spectra of ODN was developed to determine their monoisotopic molecular weights as well. Exact masses of ODN ions are determined with a 100-fold increase in accuracy relative to other available deconvolution algorithms. A computer program was developed to predict the chemical formula, exact masses, and mass-to-charge ratios of all digest fragments of ODNs. The program is compatible with most PCs and may be implemented as a Microsoft Excel add-in functionality. |
