Mass difference of -18Da

Heather Peto hp217 at cam.ac.uk
Fri Apr 27 08:13:21 EST 2001


Hi Dr Evdokimov

The freshly dissolved peptide does not give the same two peaks, I haven't
tried varying the
pH of injection. (Being new to mass spec, how does that affect the spectrum?
Its a MALDI I am running.)

The sample was kept for 1 month at 60oCentigrade and pH 3, 20mM phosphate
buffer, with 10% D2O,
The peptide was orginally purified by reverse phase chromatography so there
is probably some TFA around. The sample has not seen sunlight much.

> Methionine oxidation
>would also give you extra peaks - i.e. when the not-so-stable selenoxide
>undergoes further oxidation and decomposition. Do you have any other
>peaks in your MS ?

I see some degredation products, a small amount of the peptide has been
cleaved, I am currently working out the fragments from their mass. (Its an
15N labelled sample so its not easy to put the mass fragments into the
computer program I use on the web, Find Pept at Expasy .) Thus far all the
possible cleavage fragments do not occcur around the Methionine. Sequence:
SLRRSSCFGGRMDRIGAQSGLGCNSFRY
we are doing 15N dynamic studies on the peptide.


>-18 does sound like loss of water - and it can happen via several
>mechanisms, not just cyclisation of the ends.
My peptide chemistry is quite bad, what other mechanisms are there? Where is
a good place to look these up?

many thanks for all your help

Heather :)






"Dr. Artem Evdokimov" wrote:

> Hi,
>
> Did you run the mass spectrum on a freshly dissolved peptide, maybe
> injected under neutral conditions ? Does it show the same two peaks ?
>
> Could you elaborate a bit more on your solvent system, type of acid and
> the pH/temperature of storage. Also was the sample exposed to sunlight
> for extended periods of time ?
>
> +18 could be plus one fluoride, minus one hydrogen (is there TFA present
> ?)
> it also could be a tightly bound water molecule. Methionine oxidation
> would also give you extra peaks - i.e. when the not-so-stable selenoxide
> undergoes further oxidation and decomposition. Do you have any other
> peaks in your MS ?
>
> -18 does sound like loss of water - and it can happen via several
> mechanisms, not just cyclisation of the ends.
>
> Heather Peto wrote:
> >
> > Hi
> >
> > I have a peptide of 28 AA's. After leaving it in acidic conditions for a
> > week I run the mass spect and a proportion
> > gains a mass of 18Da (I assume that to be either Methionine oxidation
> > (+16Da, (but where are the other two Daltons?) or the addition of water
> > chemically, but where and how?),
> >
> > But interestingly I also get a significant peek with -18Da!
> > What could this -18 be? One guess of my own is that it is a cyclisation
> > of the N-and C-termini with the by product of water. (How could I test
> > this?, does the low pH (pH3) facilitate this?)
> > Any other guesses? The +18 and -18 peaks are roughly the same size.
> >
> > Heather :)
>
> --
> |Dr. Artem Evdokimov   Protein Engineering |
> | NCI-Frederick        Tel. (301)846-5401  |
> |              FAX (301)846-7148           |
> |        eudokima at mail.ncifcrf.gov         |
> |      http://www.ncifcrf.gov/plague       |




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