Hi I have hydrolysed/digested some genomic DNA into single nucleosides and I am interested to see the amount of each individual nucleosides in the samples using LC/MS. However we have run into the problem of the peaks in the samples not matching the retention time of the commercially bought nucleoside standards, hence difficult to validate the individual identity of the peaks.
we think the main problem lies in the DNA samples were in the digestion buffer while the standards were run with the HPLC mobile solvent (acetatenitrate) that will constitute the differences of the retention time. Does anyone have any suggestion on how to process the DNA samples, like whether I should dry them first so that the dried DNA can be resuspended in the solvent?
Was the DNA digested to nucleosides (no phosphate) or nucleoside monophosphates (nucleotides), and which form were the standards? Are you sure the DNA was digested completely to mononucleosides (or mononucleotides)?
Hi Ka Hou,
I am not sure why you have different retention time between digested Nucleosides and standards. However, I have separated nucleosides using HPLC before and I used a different solvent (methanol). I did not have differences in retention time between samples and standards, so your hunch about the digestion buffer may be right!
For reference, here is the paper published a few years back when I was lucky enouh to work in the lab of J.K. Blusztajn at Boston University School of Medicine: http://www.ncbi.nlm.nih.gov/pubmed/17724018 (free access)
The HPLC method originated from an older Paper by Gehrke et al.: http://www.ncbi.nlm.nih.gov/pubmed/6209294 On pubmed there is only the abstract, but I think you can get the whole paper on the journal homepage: http://www.sciencedirect.com/science/article/pii/S0021967301891895 (if you can't get it contact me and I will send you a copy)
Also, I have one more idea on what may be the problem: what did you digest your DNA with (we used first nuclease P1 and then alkaline phosphatase, see references)? And how did you check for successful (i.e. complete) digestion? [edit] sorry Adam Shapiro, I did not see your post. This idea is of course pretty much exactly what Adam suggested! [/edit]
Hope this helps & good luck!
Nick
It is possible that you have modified nucleosides, e.g. methyldeoxycytidine or deoxyinosine. It is also possible that you have both nucleosides and some nucleotides, or even the bases - it depends on the hydrolysis method. And of course you should be sure that hydrolysis was complete.
Hi all, the DNA is supposingly being hydrolysed completely to single nucleosides using method from Quinlivan & Gregory 2008 http://www.ncbi.nlm.nih.gov/pubmed/18718928, although I am not entirely sure this is the best method (I am using it because it's convenient and some other good papers have cited using this method). The enzymes involved are Bezonase (works like Nuclease P1), alkaline phosphatase and phosphodiesterase I all in 1 mix and incubate at 37C for 6 h.
I have used the nucleosides standards (A, C G, U, dA, dC, dG, T). To be absolutely sure I will also include the standards for all the NMPs (AMP, CMP, GMP, UMP etc) in the future to see if the digestions have been completed. Do you all have any other suggestions to check for complete hydrolysisof the genomic DNA?
Having said that the M/z ratio does correspond to the standards though, just not the retention time, making me wonder the problem might lie on the buffer the DNA is in.
Nick might I ask when you was doing it, was the DNA resuspend in methanol too upon injection into the HPLC? I do realise that many people use methanol as solvent so maybe that could also be another problem. (I am relying on the mass spec facility here to run the samples as I have no prior knowledge of MS, and they have been using acetonitrile with small % of formic acid as the mobile solvent as a standard practice).
Just a quick note from what I can remember: The DNA was not suspended in Methanol. It was in H2O before digesting with nuclease P1 and AP. We added the enzymes and digested (first P1, then AP - not at the same time), then continued straight to HPLC (As far as I remember we did not remove the enzymes before HPLC.)
I am not an expert in this field, but I would guess that the mobile phase is your problem. Maybe you need to play with the pH or switch to methanol (or both). If you check the Gehrke publicaiton it is explained nicely there (great publication although it looks quite old meanwhile).
Hi Nick, is it possible for you to send me the Gehrke's paper here? I have tried to get access to it without any lucks.
many thanks in advance!
Sure :-) I don't think there is a way to share files gere in ResearchGate, but if you follow me back we can exchange email addresses and I'll send you a pdf.
Hey Nick Thanks! I have managed to obtain the paper. Thanks everyone for all the help. I have dried the DNA and RNA and will resuspend them in the mobile solvent before subjecting them to LC/MS and see whether the retention time will improve.
Many thanks everyone for all the contributions.
You are welcome :-) It would be great if you could post here to tell us if it worked.
Good luck!
Nick