Mass difference of -18Da

Dr. Artem Evdokimov eudokima at mail.ncifcrf.gov
Fri Apr 27 09:28:36 EST 2001


I may not be the best expert on this but I could try to provide a few

> 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.)

For MALDI not as much as for ESI-MS, however the pH would affect the
rate of desorption to some extent because the charge state of the
molecule would be different. What's more important is that pH would
change the local chemistry of the solution - even though MALDI is a very
gentle method of desorption, it can produce artifacts and breakdown
products. Depending on the detection delay, you can even make breakdown
products to be your main analyte.

> The sample was kept for 1 month at 60oCentigrade and pH 3, 20mM phosphate
> buffer, with 10% D2O,

Do you see any sizeable % exchange of deuteriums, then ? Does the +-18
mass take into account your isotopic composition ?

> The peptide was orginally purified by reverse phase chromatography so there
> is probably some TFA around. The sample has not seen sunlight much.

Probably came in as TFA salt.

> 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:

Hmm. What else can happen...

You only have two carboxyls on this whole peptide - the D residue and
the C-terminus. You have one amine (the N-terminus) and 5 guanidines (R
residues). Intramolecular cyclization of amines and/or guanidines with
carboxy groups can and does happen but it would not be very fast at pH
3. If you get oxidation of the peptide, you lose 2 A.U. on the C-C bond
(minus two hydrogens). Oxidation of  would indeed give +16, however you
can also gain 17, if you gain two oxygens and lose the methyl in case of
demethylating oxidation. (+32 and -15 = +17).
You can hydrolyze the Q and N amides but the rate of this would be close
to the rate of spontaneous hydrolysis of peptide bond. main product of
hydrolysis would be the free acid which would mean that you lose 15+2
(if you count 100% N-15) and gain 16+1 with net change of zero.

The loss of 18 sounds like some kind of loss of water molecule - and it
can be some kind of intramolecular cyclization - either into a lactone,
lactame or some other kind of heterocycle, as long as the local geometry
allows it. I am sorry that I can't be of more help but so far these are
my best guesses. Additional possibility could be slow decomposition of
the arginine - it can for instance lose imidate and form an ornithine.
Unfortunately, loss of imidate is too much - 14*2 (or 15*2) plus 12 plus

I have just spoken to a couple of our peptide wizards and they also
aren't quite sure what could cause the loss of 18. If you ever figure it
out definitively, I certainly would like to know :)

|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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