I think you understand the limitations and what is possible. I don't understand why exactly this AA would fragment the way you are describing, but it is not something we have attempted to support.

One other option is to use the "Special ions" in Transition Settings - Filter and define the two ions you seem to want to target there. Not sure that is any better, but it is another way to target explicit fragment ions that are not well supported by other Skyline settings.

Glad you found a workaround. Clearly we were not aware of this case before your post. So, thanks for posting here.

--Brendan

Thanks, Brendan. I will give special ions a try. The background of why I was looking for these custom fragments is that they are present in ECD/ETD spectra of only iso-aspartate-containing peptides whereas aspartate-containing peptides show only regular c and z ions. ECD also shows a fragmentation pattern that can distinguish Leucine and Isoleucine. I have added a couple of references in case they can be of any help.

doi.org/10.1007/s13361-017-1674-3 An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides
doi.org/10.1007/s13361-016-1487-9 Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

--Ankit