We usually derivatize PVDF-bound samples with acrylamide (Aldrich) and
have not seen aberrant cleavages at PAM-Cys with Lys-C, but do see the
internal PAM-Cys.
As a side note, it seems like about a third of the Cys in a protein gets
derivatized by acrylamide when using Novex gels. When you alkylate with
something other than acrylamide as part of a digestion protocol, the Cys-
containing peptides will be split between peaks containing PAM-Cys (which
tends to make peptides elute later than other derivatives) and peaks
containing Cys derivatized with the other alkylating agent. In the end, the
effective yields of the Cys peptides go down, unless you alkylate with
acrylamide.
To bad Novex doesn't bump up the free acrylamide in their gels a little
more. Then we would never have to alkylate Cys. Gee, I wonder where the Cys
derivative with the crosslinker elutes on a sequencer?
Steve
====================
Stephen Tindall
Argo BioAnalytica, Inc.
Phone: 1-973-605-2100
Fax: 1-973-605-2104
StvTindall@aol.com
====================
Subj: Non-specific cleavage for endoproteinase lys-c
Date: 98-02-25 11:30:28 EST
From: fernaj@rockvax.rockefeller.edu (Joseph Fernandez)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Over the past 3 years we have observed on two occasions possible cleavage at
carboxyamidomethyl cysteine (CAMC) using endoproteinase lys-c from
achromobacter. Last night we sequenced a peptide obtained after digestion of
a PVDF-bound protein (after reduction and carboxyamidomethylation) that was
found in the database using the BLASTp search program. It ended up with a
cysteine residue before the first residue thus indicating a camc clip. Prior
to these three years we did not routinely reduce and alkylate cysteines and
never observed a cys clip before. The first two times we could never
definitively prove the CAMC clip, but this one has a lot of evidense. Here
are my questions 1) Has anyone observed this non-specific cleavage before.
2) The R/A we perform is complete for most proteins (we have never observed a
peptide with a free cysteine since performing R/A) so most likely we have
alkylation. Another possibilty is the cys has been modified the acrylamide
cysteine and that is what is cleaving (although there were 2 CAMC's
observed in the peptide). What does anyone think of this?
3) The amino terminal residue in the sequenced peptide was HIS. Anyone ever
observe cleavage at the N-terminal side of HIS before? I do not recall our
earlier runs if there was a HIS or not.
4) Anyone know of any standard proteins low in lysines that could have a CYS-
HIS bond to check out the non-specificity?
5) One last thing is that WAKO's literature states that the enzyme cleaves at
S-aminoethylcysteine as well as lysine. Anyone know of a side reaction of
CAMC to form S-aminoethylcysteine or more likely acrylamide cysteine to
generate into S-aminoethylcysteine? Is there such a thing as ACRYLAMINE in
acrylamide?
Looking foreward to any answers, suggestions, opinions or useful or humerous
comments.
Joe
3)
Joseph Fernandez
Associate Director
The Rockefeller university
Protein/DNA Technology Center
1230 York Ave. New York, NY 10021
Phone: (212)-327-8869
FAX : (212)-327-8620
email: fernaj@rockvax.rockefeller.edu
Lab Web Page: http:\\pdtc.rockefeller.edu