Dr. Perez,
Although not real precise, you could stop the digestion reactions by
freezing in liquid N2 and vacuum drying the sample/aliquots at different
time points. These aliquots could then be resuspended and spotted for MALDI
at a later point in time.
Alternatively, you could use a concentration-dependent digestion vs. a
time-dependent digestion. These carboxypeptidase digest experiments could
be performed directly on the sample stage, dried and submitted for MALDI.
Concentration Dependent CP-Y:
Patterson, Tarr, Regnier, Martin, Anal. Chem., 1995, 67 3971-3978.
Time dependent CP-Y:
Wooods, Huang, Cotter, Pasternack, Pardoll, Jaffee, Anal. Biochem. 1995,
226, 15-25.
Best regards,
Lloyd
Lloyd W. Sumner, Ph.D.
Head, Biological Mass Spectrometry
The Samuel Roberts Noble Foundation
PO Box 2180
2510 Sam Noble Parkway
Ardmore, OK 73402
Voice 580-221-7392
Fax 580-221-7380
Email lwsumner@Noble.Org
-----Original Message-----
From: J.Manuel Perez [mailto:perez@helix.mgh.harvard.edu]
Sent: Monday, August 14, 2000 5:30 PM
To: Recipients of ABRF List
Subject: C-terminal sequencing
Dear ABRF people:
I am interested in doing C-terminal sequencing using Carboxypeptidase Y
(CP-Y) of C-terminal amide peptides and we are using bombesin
(pyro-EQRLGNQWAVGLM-amide) as a model peptide to help us develop an assay. I
notice that most people stop the reaction simply by adding MALDI matrix
solution (alpha -cyano-4-hydroxycinnamic acid in 1:1 acetonitrile/0.1% TFA)
at different time points to generate the sequencing ladder before running
the samples in the MALDI.
Is there any other way to stop the reaction besides adding the matrix
solution? I will be just doing the enzyme digestion and supplying the
samples to a core facility for them to run the MALDI (they will then add the
matrix solution). I tried adding 0.1% TFA and that did not stop the
reaction. The 1-minutes time point looks like the 3, 5, 10 and 15-minutes
time points.
By the way - it looks like CP-Y digested the last six amino acid of this
C-terminal amide peptide with no problem.
Thank you very much in advance
J. Manuel Perez, Ph.D.
Center For Molecular Imaging Research
Massachusetts General Hospital
Boston MA
(617)-726-5788
(617)-726-5789
Fax (617) 726-5799
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