Team:Vanderbilt/Experiments/LCMS
Mass-Spectroscopy
Methods for precise DNA oxidation
We adjusted our antibody-labeling protocol to make it possible to extend this technique to oxidized DNA samples. Although we were unable to match the precision of the irradiation experiments, oxidation-to-signal linearity measurements showed a fair association. However, because the effect sizes we were anticipating were within the range of the ambiguity, further protocol optimization is needed before it can be applied to different oxidized samples.
Quantification of oxoguanine levels in synthetic DNA samples
Liquid chromatography--mass spectrometry (LC-MS) is the current standard for measuring DNA oxidation due to its high sensitivity and specificity. We experimented with methods to analyze levels of oxidation in our gene samples, and achieved reliable detection of hydrolyzed bases. We could detect 8-oxoguanine (8oG) signal in our analytical standard, and characterized four discrete protocols for in vitro oxidation at a range of doses (hydrogen peroxide, methylene blue/light, UV irradiation, Mg). Although signal was often low, we have begun to compare our optimized sequences with improved oxidation methods.
Our preliminary results show a correlation between exposure to peroxide oxidant and the quantified peak area for 8oG. However, our data on oxidation-optimized synthetic DNA sequences could not be quantified because the signal we detected was below the threshold of 10^4 intensity units, which is a standard in the field above which signal can be reliably distinguished from noise. Because we demonstrated that LC-MS detection of oxoguanine is proceeding correctly, we are pursuing alternative methods of hydrolyzing and purifying the nucleotides of our synthetic DNA samples.
