Axel
Dr. Ming F. Tam wrote:
> Dear Protein Gurus:
>
> We have isolated an unknown protein from a mixture on reversed-phase
> column. It is well separated from other contaminants and online LC/MS
> told us that it has a MW of 26540 and a minor component (about 5 to 10%=
) of
> 26597, a difference of 57 Da. The protein starts with an N-acetylated
> serine, we can obtain Edman results after mild TFA treatment. We
> carboxymethylated then digested the protein and sequenced all the
> fragments. We obtained 26597 for the sum of all these fragments (minus=
the
> carboxymethyl groups). We have digested the protein with Lys-C, Arg-C =
and
> Asp-N. In addition to the mass data we always sequence the first 2-3
> residues from each peptide to confirm that we are looking at the right
> fragment. We always come up with the number 26597 after adding up the
> fragments and cannot detect any fragment that doesnot fit the sequence
> data. We have also tried dissolving the digests in guanidine-HCl and
> running the column at 60=B0C as suggested recently for hydrophobic pept=
ides.
> We could not detect anything unusual either.
>
> Are there any modified amino acid that has an addition of 57 Da and elu=
tes
> off the Procite sequencer at the position of a regular PTH-AA?? What o=
ther
> explanation we have?? Any suggestion is most appreciated.
>
> Ming
>
> *********************************
> Ming F. Tam
> Institute Molecular Biology
> Academia Sinica
> mbr405@ccvax.sinica.edu.tw
> *********************************
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Axel Ducret, Ph.D.
Senior Research Biologist
Merck-Frosst Canada Inc.
Dept. Biochemistry and Molecular Biology
P.O. Box 1005
Pointe-Claire-Dorval PQ H9R 4P8
Canada
tel. + (514) 428-3428
fax + (514) 428-4900