I am using an oligopeptide substrate as a substrate for my protein which I believe cleaves all amino acid residues from the oligopeptides of 3-10 residues long. My issue here is to prove that all residues are being cleaved from the substrate. Is there an assay to identify these different residues to prove the point. I know of some basic tests in biochemistry but I think they aren't that sensitive. can anyone please suggest something easy and sensitive to do this here?
Hi there,
If I understand well your protein degrades the peptide substrate completely into aminoacids. If this is the case, aminoacid analysis of the reaction mixture should allow to detect the apparition of free aminoacids.
Some doubts in your question but I would recommend to talk to an expert in Mass spectrometry that you may probably find in your own Institution.
I would suggest amino acid analysis, if the substrate in your reacton is at high concentration. otherwise mass spectrometry is a good way to detect targets with high sensitivity. You could use the reacted mixture as sample for MALDI_TOF MS analysis, then check the masses in the spectrum and match them with your peptide sequence to see whether all amino acids were cleaved or not. after removing the enzyme.
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