I am trying to use N-terminal peptides for quantitation. The initiator met is cleaved off cotranslationally for many proteins and, as a result, the peptide is not properly recognized as a tryptic peptide by Skyline (since it is not preceded by a K or R but by M) if we digest the proteome with trypsin [KR | P]. When digesting the proteome with trypsin-CNBr [KRM | P] then cleavage also occurs at internal M and the corresponding peptides are incorrectly marked as having a missed cleavage. Is there any way to define a digestion scheme that is based on trypsin [KR | P] but also includes cleavage of the initiator methionine, which is common for many proteins? My lab is working on quantifying the N-terminal peptide because we study effects of enivronmental stress on N-terminal modification (acetylation, etc) of proteins.
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Dietmar