From: Amina Sarah Woods <amina@welchlink.welch.jhu.edu>
Subject: RE: CPY & MALDI
Date: Tue, 21 Jan 1997 12:20:40 -0500
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Lisa:
we do lots of CPY digests in our lab both on the MALDI probe and in
solution.
On the probe:
Deposit 0.3 ul substrate followed by 0.3 - 0.6 ul of a 50 mM ammonium
citrate buffer pH 6.5 followed by 0.3 ul enzyme (0.05-0.3 ug/ml) incubate
at RT for 15-120 sec. Stop the reaction by adding 0.3 ul saturated alpha
cyano-4-hydroxy-cinnamic acid in 50% ethanol.
Although CPY is supposed not to cleave K and R, sometime the enzyme does
it quite efficiently depending on the peptide. I usually do 2 to 3
digests. My best result has been 7 residues cleaved, but sometimes it
stops after 2 or 3. The success of the digest pretty much depends on the
amino acid composition of the peptide. If you have any question please
give a call. As to the size of the peptide we've been able to cleave from
the carboxyterminus of as little as 630 amu and as large as 18,000. We
have published this method in an article in Cell (Cell, vol. 79 649-658 Nov
18, 1994), and Analytical Biochemistry (Anal. Biochem. 226, 15-25 Jan
1995).
Amina S. Woods Ph.D.
Johns Hopkins School of Medicine
(410) 614-4981
amina@welchlink.welch.jhu.edu
----------
From: bibbs@scripps.edu (Lisa Bibbs)
Sent: Saturday, January 20, 1996 11:03 AM
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Subject: MS: CPY & MALDI
Hello Mass Spec people
My colleague has asked me to pose the following questions to you.
Thanks in advance for info.
Lisa
>Mime-Version: 1.0
>Date: Fri, 17 Jan 1997 14:54:47 -0800
>To: bibbs@riscsm.scripps.edu
>From: bothner@riscsm.scripps.edu (brian)
>Subject: question
>
>Hello Lisa
> Here is the question I have: Does anyone have experience with peptide
>sequencing using CPY and MALDI/TOF? I would like to know what size
>peptides work well (how large)? How many residues are you typically able
>to sequence using multiple dilutions of the CPY?
> Thank you, Brian
>
.