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 t=
he
carboxymethyl groups). We have digested the protein with Lys-C, Arg-C an=
d
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 peptid=
es.
We could not detect anything unusual either.
Are there any modified amino acid that has an addition of 57 Da and elute=
s
off the Procite sequencer at the position of a regular PTH-AA?? What oth=
er
explanation we have?? Any suggestion is most appreciated.
Ming
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Ming F. Tam
Institute Molecular Biology
Academia Sinica
mbr405@ccvax.sinica.edu.tw
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