A common question we hear is “Do I need to define a modification for protein C-terminal Lysine clipping?” The answer is yes and no, depending on how you would like to report the Lysine clip.
By default, Byonic searches for single residue clips from N- and C-terminus of all protein chains. If the user doesn’t define any modifications and perform a fully-specific search, Byonic would identify both clipped and unclipped terminus peptides. For example, both of these peptides would be in the results in a default fully-tryptic search:
K.SLSLSPGK.-
K.SLSLSPG.K
They will both appear as wildtype peptides. The user can create a dedicated tab in Byologic reports to see a relative abundance of the two, as follows:
In the above table, note the filter applied on “Sequence (unformatted)” column to show only the selected two peptides. (Alternatively, once can use the “IsCterm” dynamic column to filter in C-terminal peptides automatically, rather than selecting sequences manually.)
Another approach is to define the Lysine clipping as a modification:"Lys-loss / -128.094963 @ Protein CTerm K". In this method, the user also needs to turn off automatic search of single-residue clips. This is accomplished by the “cleavage_flags” command in Byonic. Simply enter the below in the custom modifications box:
% Custom modification text below
cleavage_flags=0
With this method, Lys-loss will appear as modification, the following two peptides will be identified:
K.SLSLSPGK.-
K.SLSLSPGK[-128].-
Since Lys-loss peptide is a modified peptide, the reporting will be straightforward by the default PTM report template:
Taking this a step further, if the user would also like to search for Gly-loss+Amide / -58.005479 on protein C-terminus, there are two options:
- if cleavage_flags=0 is used and Lys-loss is defined as a variable - common1 modification, then Gly-loss+Amide can be defined as a variable - rare1 mod, so that both modifications can be searched at the same time. The resulting peptide ID would then be:
K.SLSLSPG[-58]K[-128].- - If cleavage_flags=0 is not used, then the user can set Gly-loss+Amide as variable - rare1, and the result will be:
K.SLSLSPG[-58].K