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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