Difference between revisions of "Team:Vanderbilt/Experiments/LCMS"

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<h2>Mass-Spectroscopy</h2>
 
<h2>Mass-Spectroscopy</h2>
 
<h4> Summary </h4>
 
<h4> Summary </h4>
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<p>  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 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.</p>
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<p>  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.</p>
 
<p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/4/4f/Vu16_figure10.png" alt="Fig10" style="width:600px;height:200px;" /></p>
 
<p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/4/4f/Vu16_figure10.png" alt="Fig10" style="width:600px;height:200px;" /></p>
  
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<p> With regard to 8-oxoG standard detection, we were compelled to make a number of modifications to our protocol to detect our compound within a triple quadrupole mass analyzer. Xcalibur software was used to predict fragment masses that we eventually concluded could not be detected in our sample. Fortunately, a number of predictive modifications to our model demonstrated our compound could be detected in our sample and uncovered the corresponding elution time. <br>
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<p> 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. <br>
 
<p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/a/a6/Vu16_figure11.png" alt="Fig11" style="width:600px;height:200px;" /></p>
 
<p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/a/a6/Vu16_figure11.png" alt="Fig11" style="width:600px;height:200px;" /></p>
  
 
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Revision as of 08:23, 27 November 2016

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Mass-Spectroscopy

Summary

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.

<img src="Vu16_figure10.png" alt="Fig10" style="width:600px;height:200px;" />

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.
<p style="text-align:center;"><img src="Vu16_figure11.png" alt="Fig11" style="width:600px;height:200px;" />

</p>

</html>