How to import amino acid substitutions from ProteinPilot 5.0 search results

How to import amino acid substitutions from ProteinPilot 5.0 search results christof.lenz  2020-06-16

Dear all,

I am looking for a systematic way to import peptide search results from SCIEX ProteinPilot 5.0 including amino acid substitutions. In 'thorough' mode, ProteinPilot allows to search peptide sequences including all amino acid substitutions. Scoring is then adjusted using an underlying BLOSUM substitution matrix and goes into scoring, so we could use a peptide score cutoff here.

I am stumped, however, on how to import the search results including the 180 or so possible AA substitutions into Skyline to look for these peptides e.g. in HR-MS1 or DIA data. Is there any good way to do this, except 'declaring' each potential substitution as a variable PTM?
Data are from a SCIEX TT5600+ with Analyst TF 1.7; PP is version 5.0 rev. 4769; Skyline is on the latest version.

Any hints or experiences much appreciated!!


Brendan MacLean responded:  2020-06-16

Hi Chris,
I assume the search results contain the actual amino acids in the matched peptides? i.e. if a substitution occurs, the search results report the matched peptide as containing the substituted amino acid residue. e.g. The FASTA lists:


But the search engine finds:


If this is the case, then you can add these peptides as targets using View > Spectral Libraries, selecting one, and clicking the Add button. Or clicking the Add All button.

I don't have an easy way to give you protein associations for these peptides which do not appear in the FASTA sequences, but this is a relatively easy way to get them into the Skyline Targets list.

Otherwise, you could also make your own list of matched peptide sequences with protein name associations in Excel and add them in Skyline through Edit > Insert > Peptides.

That's about all I can think of. Hope one of these is helpful to you in this case.