Possible RNA Degradation in Mass Spec?

[RNAguy123]

Hey all,

 

So I have a sample of RNA with a MW of ~7000 g/mol forming a stem-loop structure, and about a month ago I used ESI to run the sample on the mass spec and saw the expected distribution of charge states cleanly (sprayed from methanol:water in the negative mode). I stored the RNA sample in MQ water at -20 celsuis in the meantime. Now I tried again today using the same ESI conditions with my sample and saw nothing new to the background except for a peak at m/z=264 or 265. There is nothing in the upper ranges of m/z=600-1000 which I was looking successfully at a month ago. Now my first guess is the RNA has degraded during storage, but what I don't get is wouldn't I see a distribution of fragments and not just a single clean peak at m/z=264? The peak was not present in the solvent background alone so I have no idea what it could be.

 

Any insight would be much appreciated! Thanks a lot

[CharonY]

One month in water is certainly not something that most RNA would survive. Is it double-charged? It definitely seems to be degradation and there is a chance that your ionization may fragment whatever is left there. Do you do direct injection or do you have some level of separation?

[RNAguy123]

Thanks very much for your reply. I agree I do think it is degradation as well and my fault for improper storage, I just don't get the resulting mass spec, shouldn't I see an array of fragments for degraded RNA and not one sharp peak at m/z=265? My sample can adopt a maximum charge of -24, however unlikely to see that high of a charge state following ESI. I do direct injection and then can separate afterward depending on m/z. Again I do agree with you in thinking it is degradation, I am just trying to make sense of the resulting mass spectrum.

 

Thanks again CharonY

[CharonY]

My points were that a) for a double charged ion you are in the range of individual nucleotides and b) you could have in-source fragmentation and, especially in case of direct injection of low concentrated sample you may have additional suppression effects. Assuming that all is degraded potential degradation products may not survive ionization and/or have bad s/n. E.g. the only strong signals may be final fragmentation products. It is clearly only a guess, though. Running a gel could provide you some insights whether there was total degradation pre-MS.

[BabcockHall]

Did you store frozen, at refrigerator temperature, or at room temperature? From your initial post, it sounds as if you stored it frozen. My guess is that this is better than leaving it at room temperature, but I might have quick frozen the sample and stored it at -80 °C unless I had a better protocol. I cannot speak to the specific question of the best way to store RNA, but I can make some very general comments about freezing. One is that the pH of a buffer solution can change as the solution cools, and RNA is labile in basic conditions. Two is that there are shearing forces during the freezing process which can cause problems in some instances. Therefore, I would look into the use of cryoprotectants and fast freezing protocols for RNA. I do not have an explanation for the mass that you are seeing.

[RNAguy123]

Did you store frozen, at refrigerator temperature, or at room temperature? From your initial post, it sounds as if you stored it frozen. My guess is that this is better than leaving it at room temperature, but I might have quick frozen the sample and stored it at -80 °C unless I had a better protocol. I cannot speak to the specific question of the best way to store RNA, but I can make some very general comments about freezing. One is that the pH of a buffer solution can change as the solution cools, and RNA is labile in basic conditions. Two is that there are shearing forces during the freezing process which can cause problems in some instances. Therefore, I would look into the use of cryoprotectants and fast freezing protocols for RNA. I do not have an explanation for the mass that you are seeing.

 

Thanks a lot for the suggestions. I will look into cryoprotectants and definitely will use buffer solutions now. I appreciate the insight!

My points were that a) for a double charged ion you are in the range of individual nucleotides and b) you could have in-source fragmentation and, especially in case of direct injection of low concentrated sample you may have additional suppression effects. Assuming that all is degraded potential degradation products may not survive ionization and/or have bad s/n. E.g. the only strong signals may be final fragmentation products. It is clearly only a guess, though. Running a gel could provide you some insights whether there was total degradation pre-MS.

 

Got it CharonY, thanks a lot it makes sense. I will definitely run a gel and see what sort of state my sample is in, thanks for the suggestion.

[CharonY]

Did you store frozen, at refrigerator temperature, or at room temperature? From your initial post, it sounds as if you stored it frozen. My guess is that this is better than leaving it at room temperature, but I might have quick frozen the sample and stored it at -80 °C unless I had a better protocol. I cannot speak to the specific question of the best way to store RNA, but I can make some very general comments about freezing. One is that the pH of a buffer solution can change as the solution cools, and RNA is labile in basic conditions. Two is that there are shearing forces during the freezing process which can cause problems in some instances. Therefore, I would look into the use of cryoprotectants and fast freezing protocols for RNA. I do not have an explanation for the mass that you are seeing.

 

I found that a key component is also purity of chemicals. Contamination with metals or, even worse, RNAses can result in rapid degradation. Even flash freezing does not always help, as the thawing process can be an issue. At the same time, an extremely clean sample that is devoid of any RNAses can be surprisingly stable even at -20C. Also note that nucleic acids are most stable at slightly alkaline conditions (ca. pH 8). Freezing itself seems to be more problematic in larger nucleic acids, but storing in aliquots is generally a good strategy. Finally, if one considers storing for years, a precipitate may be worthwhile. Other than that one should add that quite a bit of lab-vodoo is also involved.

Edited July 19, 2017 by CharonY

[BabcockHall]

As one raises pH, transphosphorylation becomes more of an issue. What happens at low pH, loss (or perhaps epimerization) of glycosidic bonds?

Edited July 20, 2017 by BabcockHall

[CharonY]

At acidic condition depurination starts to occur. At high pH 2'-OH deprotonation is an issue, resulting in hydrolysis.

[RNAguy123]

Thanks again for all the replies. I am going to provide an update.

I ran a 15% PAGE in duplicate with my sample, and two distinct bands showed up right as expected for a 22mer of this size indicating degradation is not the problem (or at least the sample is not yet degraded so perhaps it was at the source, although two weeks prior I ran the sample with identical conditions and had great results).

So I guess I remain baffled.

[CharonY]

Why did two bands show up? But assuming it is still alright (In my initial response I thought that you had a total RNA extract, but upon re-reading it would not make  a lot of sense for conformational studies), the issue seems to shift towards the analytical side of things.

[RNAguy123]

Sorry I was unclear, I meant one band for each run (since I did it in duplicate there were 2 bands total). But each loaded lane had just one distinct band corresponding to a 22mer. My sample is a desalted RNA oligo from Sigma, I'm just trying to make sense of the detreioration of signal in the mass spec.

 

Thanks again CharonY

[CharonY]

Well, those are far more stable. The next thing is probably doing a run with a reference sample on the MS and doing a re-run.