A secondary amine at the N-terminus can cause the cleavage of the first
AA during the Edman coupling steps and you will see the second AA of the
sequence in the first Edman cycle. This is probably what you are seeing.
You see the same thing for N-termini modified with vinyl pyridine,
acrylamide, etc.
Steve
====================
Stephen Tindall
Argo BioAnalytica, Inc.
====================
Subj: ProtSeq, pegylated peptides
Date: 4/29/99 12:05:05 PM Eastern Daylight Time
From: vernon.shoup@regpha.com (VERNON SHOUP)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular
Resource Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
It's my turn to accidentally send a non-ABRF message out to the ABRF
Rountable. I was going to gather a bit more information before sending out a
question to the group!
Anyway, we Pegylated a recombinant protein, using chemistry that attached
20K PEG to the free amine groups of lysine and the N-terminus. The linkage
contains a secondary amine. We then did tryptic mapping, and found a
distinct new peak, as discussed below (and in the previous message).
We isolated and sequenced the new peak (152') observed in the tryptic map of
this material. The odd thing was that the main sequence observed started at
the SECOND amino acid of the N-terminal peptide, i.e. FTEH....
(The first amino acid was an alanine.) From a naive point of view, this
peptide should have been blocked from participating in the sequencing
reactions.
Does anyone have any insight about how this could come about?
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12144
vernon.shoup@regpha.com