I am finding a number of peptides in brain that are truncated at either the N-terminus or C-terminus or both compared to the canonical sequence. What is the best way to determine if these are an artifact of the prep (homogenization and ultracentrifugation) or if these are endogenous/biologically relevant?
I am doing top-down proteomics so no digestion is involved.
My thoughts for the C-terminus were to do the entire prep in 18H2O if the truncations at the C-terminus are real/biological then no 18O2 should be incorporated at the C-term. -- Thoughts?
Any thoughts on the N-term?
Hi Norelle,
The intact protein can be derivatized/labeled to contain a Dimethyl group. During this reaction, some of the epsilon side chain of the internal lysines may also be derivatized ; in general, the N-terminus is the main amino acid that gets dimetyhlated.
If the N-terminal amino acid is "blocked", then you will not be successful in derivatization of the the N-terminus.
I have attached a recent publication on this topic.
Best,
Hediye.
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