Was there any urea present in your digest? Any cyanate derived from
urea breakdown will readily block alpha-amino groups. And the urea
doesn't even have to be added to the digest for this to happen;
incomplete removal of urea during the preparation of the protein to be
digested can eventually lead to blocked N-termini. If you believe that
this is the cause of your problem, you might want to add methylamine to
20 mM in the digest to scavenge cyanate.
Mike Klein
Amgen, Inc.
> ----------
> From: Conti Amedeo[SMTP:conti@ch.unito.it]
> Sent: Tuesday, April 21, 1998 4:46 AM
> To: Recipients of ABRF List
> Subject: block pep
>
> Dear ABRFers
> we have oxidized a 9KD protein containing 8 Cys (4 S-S) and digested
> with
> Asp-N in Tris-HCl pH 8; separation of peptides by RP-HPLC in ACN/TFA
> was
> quite good but all resulted N-terminally blocked except one starting
> with
> Cys-Cys- that gave two cicles blank and then the right expected
> sequence;
> MW of this peptide measured by ESI-MS was consistent with the presence
> of
> one cystine residue.
> Do you have any experience on which kind of blocking reaction could
> happen
> to the N-terminal cysteine (cysteic acid)?
> Thanks in advance
> Amedeo Conti
>