Prepare a 1:1 mixture (in 10 mM sodium citrate pH 5) of Carboxypeptidase Y and P(Boehringer Mannheim). Use a 1:50 weight ratio of CPP/CPY to sample. Typically prepare multiple tubes for a time course of digestion and add matrix solution directly to a tube to quench the reaction. Alternatively, I prepare a dilution series of the carboxypeptidase mixture and add this directly to the protein/peptide samples on the MALDI slide. After 10-15 min then add the matrix to these digestion mixtures.
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Gordon Alton, PhD
Gene and Protein Discovery Group
Signal Pharmaceuticals Inc.
5555 Oberlin Drive
San Diego, CA 92121
Email: galton@signalpharm.com
Phone: 619-558-7500 x8252
Fax: 619-623-0870
WWW: http://www.signalpharm.com
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-----Original Message-----
From: Karen Toney [SMTP:karent@allelix.com]
Sent: Wednesday, June 30, 1999 12:04 PM
To: Recipients of ABRF List
Subject: peptide sequencing
Dear ABRFers,
I am trying to obtain the sequence of a 9aa peptide with an N-terminally
blocked glycine. Sample amount is not a limiting factor, so there are a
few ways we are thinking about going about this:
1)CPY digestion ladder and analyzing with MALDI-TOF. However, there is
one Lys and one Arg in the sequence and my understanding is that CPY is
not particularly fond of these residues. If this is true, are there any
other enzymes we can use? Can we add CPB to the reaction mix? What
ratios?
2)We were also thinking of having it sequenced on a C-terminal
sequencer, however there are a couple of prolines near the C-terminus.
Is there any way to sequence through these residues?
Alternatively, we would like to cleave the N-terminal glycine and
proceed with N-terminal sequencing. Any suggestions or protocols for
doing this? Will the blocked N-terminal pose a problem for enzymatic
cleavage?
A final point for consideration: the peptide also contains an Acm
protected Cys residue.
I look forward to any suggestions/comments. Thanks in advance.
Karen Toney
Allelix Biopharmaceuticals