Can you see DNA damage in a Q extactive off a C18 column?

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Jen_G
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Can you see DNA damage in a Q extactive off a C18 column?

Postby Jen_G » Mon Apr 01, 2013 2:54 pm

I am throwing this question out there. I am wondering if I could detected damaged nucleotides extracted from cells treated with mutagens in the mass spec? Is it like PTMs in proteomics, where you have to enrich PTMs from the proteome in order to reliably see them by mass spec? I have no experience and would like to see if it is a feasible project.

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Doug
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Postby Doug » Sun Apr 07, 2013 6:56 pm

I do believe that you can separate DNA using C18 columns but the mobile and stationary phases would probably need to be slightly different from those used to separate proteins and peptides. I am not sure exactly how you will design your experiment but think the ionization (preferences for certain lengths of oligos), fragmentation (you may need very even fragmentation patterns for sequencing), data interpretation, and sample complexity (depending on your experiment design you could have orders of magnitude more analytes) are going to be bigger problems. In most cases LCMS is not going to be your best option to analyze oligonucleotides. But it can be done (and has been) and I am sure there are reasons to do so.
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Kelstrup
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Postby Kelstrup » Mon Apr 08, 2013 1:27 am

I've found it very hard to interpret the fragmentation spectra of DNA compared to peptides. Loads of repeated subunits having the same mass. YMMV :)

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Postby Jen_G » Wed Apr 10, 2013 5:00 pm

Thanks Doug for the reply. I want to measure 8OHdG in genomic DNA. From the literature that I have read it seems that detection is a big issue. I think that I am going to use antibody that has been shown to be more sensitive.

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Postby Jen_G » Wed Apr 10, 2013 5:01 pm

I was going to digest the DNA with MNase to get single nucleotides but I think that for a pilot study I am going use an antibody to the primary damaged nucleotide that is generated in my system.

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Doug
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Postby Doug » Wed Apr 10, 2013 8:47 pm

I see. That's pretty cool. I know people do that with amino acids. The use acid hydrolysis (or other methods) to break down proteins into single amino acids. Then they typically just use HPLC with a UV detector to quantify the amount of each amino acid (they identify amino acids by retention time). They don't even need the mass spec. But I guess if your damaged/modified nucleotide is rare it will be very hard to see. Sounds like a cool project. Good luck.
"If we knew what we were doing it wouldn't be research." -AE

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Postby tsbatth » Mon Apr 15, 2013 8:26 am

Hmm might be interesting. I've heard of some people at Roche using MALDI to screen oligos in a HT manner, but I don't think it was for sequencing purposes, mostly QA/QC . Might want to look into it.

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Postby PhilG » Thu Apr 18, 2013 9:57 am

Analyzing nucleosides by LC-MS is possible. A company called ZymoResearch sells a product called Degradase that digests gDNA down to nucleosides. Using C18 with water/Methanol/0.1% formic acid solvent systems works nicely (we use it for 5mdC quantification). Typically SRM can be set up for high sensitivity by monitoring the loss of the sugar. A word of caution if you go the SRM route; be sure other ribo- and deoxyribonuleosides do not co-elute with the compound of interest and possibly contribute to the SRM signal (an isotopic peak of a ribonucleoside might be the same as the monoisotopic peak of the compound of interest).


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