Dear colleagues,
I will summarize here the answers I got regarding our problems with the mass spec of oligonucleotides:
THE QUESTION:
Dear mass spec users,
we're having a problem trying to identify short (7mer) oligonucleotides by
ESI-MS (PE SCIEX API150EX). The problems arise from the fact that the
oligos are dissolved in 10 mM K-phosphate buffer pH 7 (in TEAA they work
fine) so we get just a lot of noise and no oligo signal and we would like
not to pass them through reverse phase. Is there a way to get some good
mass spectra in this kind of buffers, maybe playing with the ionization
potentials or else?
THE ANSWERS
>Try to run it in negative mode.
>One possible solution if you have them available is ZipTip
>purification. Although this is the exact reverse phase that you mention
>you'd like to avoid, if your worried about dilution, the ZipTips work on
>microliter volumes. You can see a description of them on Millipore's web
>page. Alternatively, you could pack a small amount of C18 or ion exchange
>resin in a pipet tip (that's what a ZipTip is), or put a small amount of
>packing in the bottom of an eppendorf and swish things around a bit.
>Finally, addition of ammonium acetate (we use citrate for MALDI, but I don't
>think its best for esi), equal volume with an aliquot of sample, could give
>you enough ion exchange to pull some signal out. Your local Millipore rep
>might be convinced to give you a tip or two to try out if you don't have
>any.
>Use another buffer like acetate, the buffer needs to be volatile. The phosphate
>will precipitate on your interface plate. Is it in negative mode? Phosphate may
>be used, but only at very low concentration levels. Phosphates like electrons.
>Stick to volatile buffers
>Have you tried to dry and resuspend in TEAA?It is simplistic,but it may
>work.
>Dear Corrado,
>already 1 mM gives problems. I suppose you are doing the MS in negative
>mode, which gives weaker signals than positive mode, and this makes
>things worse. You could use weak anion exchange and elute the oligos
>changing pH by TEA or NH3, this would be directly compatible with
>negative mode.
CONCLUSIONS:
I have to tell you that RPHPLC obviously worked well as expected, but already a resuspension
in tryethylammonium acetate (containing a 50% acetonitrile) improved a lot the oligo signal in
negative mode, even if the noise coming from the K-phosphate does not disappear.
The origin of our question is the fact that we would really see if it's possible to see the oligos
in low salinity buffers because we're trying to study DNA-protein complexes by ESI-MS.
We're open to futher advices and discussion.
Thanks a lot to everybody
This archive was generated by hypermail 2b29 : Fri Mar 10 2000 - 09:45:23 EST