Just a wild guess.If NEM was used,there is a good possibility that you have
S-ethylsuccinimido cysteine.I get
some of that even after acid hydrolysis at 150 degrees for 1hr,along with
the expected succinyl-cysteine.At cycle 1,
this species would predominate.I was going to say cysteine
S-propionamide,but the position is wrong.And,I am
guessing since I never noticed the above product on sequencing,but by amino
acid analysis,it elutes between
Val,Met and Lys.PTC analysis would have cystine in that area,but tryptophan
would elute much later.
Matt Williamson <mkwilliamson@ucsd.edu>@aecom.yu.edu> on 10/06/2000
11:40:23 AM
Sent by: Association of Biomolecular Resource Facilities
<abrf-request@aecom.yu.edu>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
cc:
Subject: Unknown PTH amino acid
Dear Protein Sequencer Experts,
I recently sequenced a sample for someone using the Procise 494 sequencer
and got an unknown peak. The sample was run through SDS PAGE under
reducing conditions, transferred to PVDF, and stained with Coomassie Blue.
The apparent molecular weight was in the neighborhood of 6000 daltons. The
first amino acid gave the unknown peak which eluted just before PTH Trp. I
checked with Bob at PE/ABI and he checked the known amino acids. He
suggested Iodo-Tyr and
alpha,gamma di-amino buteric acid as having been seen to elute in this
vicinity.
The sample went on to give a good sequence run and we were able to show
homology to known proteins from other species. The other species had a Cys
at the corresponding amino acid position. Are there any known Cys products
that would elute in this position on the Procise system? (By-the-way, the
researcher had not done any modifications of Cys prior to running the
sample on the gel, and there was another position where I saw a
Cys-propionamide (cys side reaction witth unpolymerized acrylamide) peak
eluting after position of PTH-glu).
Any insights would be greatly appreciated.
Matt Williamson
UCSD Biology Division Protein Sequencer Facility
This archive was generated by hypermail 2b29 : Mon Oct 23 2000 - 10:23:17 EDT