Dear Manuel,
CP-Y digestion is pretty tricky, as you might have guessed, and most of all the
digestion kinetics can widely change depending on the amino acid composition.
In your particular case, what I suspect is that CP-Y chop your first 6 amino
acids at a very high rate (aliphatic amino acids are preferred substrate for
CP-Y). I would recommend that you use less CP-Y (let say, a 1/10 of your
original amount). In addition, try to stop your reaction with a more
concentrated TFA solution (1 or 5%). If your buffer is buffered by carbonate,
0.1% TFA will not be sufficient to drop the pH significantly.
Hope that it helps
Axel
"J.Manuel Perez" wrote:
> 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
-- ------------------------------------------------------------------------Axel Ducret, Ph.D. Senior Research Biologist Merck-Frosst Canada Inc. Dept. Biochemistry and Molecular Biology P.O. Box 1005 Pointe-Claire-Dorval PQ H9R 4P8 Canada
tel. + (514) 428-3428 fax + (514) 428-4900
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