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Peptides not present in Skyline document after importing FragPipe results

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Peptides not present in Skyline document after importing FragPipe results tanja ziesmann  2025-11-20 01:18
 

Dear Skyline Team,

I am currently trying to import some FragPipe results using heavy/light labelling into Skyline (25.1.0.237). The import itself, using the interact.pep.xml files, following the workflow suggested by FragPipe, works, however, when I take my result list from FragPipe to filter the Skyline document, most of those peptides are not present (i.e., out of 1189 peptides 1050 are not present). I tried setting the score threshhold really low, but that did not change the outcome.
I checked and those peptides are present in the interact.pep.xml so I'm not sure what I'm doing wrong that they are not included in the document.

Best regards,
Tanja
 
 
Nick Shulman responded:  2025-11-20 10:44
You can probably get more of those peptides into the document if you go to:
View > Libraries > Library Explorer
and then press the "Add All" button.

The newly added peptides will be added to the document in a peptide list called "Library Peptides" and you will probably be able to take a look at those peptides and figure out why they were not included when the peptide search results were originally imported.

When you do "File > Import > Peptide Search", Skyline creates a spectral library and then essentially does "File > Import > FASTA". This usually ends up missing a few of the peptides that might have been found in the peptide search because the peptides don't pass Skyline's filter criteria, which might be:
1. The peptide could not have been cleaved based on the enzyme setting on the "Digestion" tab at "Settings > Peptide Settings" or has too many missed cleavages.
2. The peptide does not pass the filter criteria on the "Filter" tab at "Settings > Peptide Settings".
3. The library spectrum does not have enough detected ions to satisfy the minimum ion count on the "Library" tab at "Settings > Transition Settings".

After you have added the missing peptides to your document you can use the "Refine > Associate Proteins" menu item to get all the peptides assorted into the appropriate Proteins.

If you do not see the peptides that you want in the Library Explorer, then the problem might be that the cutoff score that you specified when creating the spectral library was too strict. However, it sounds like you did use a lenient cutoff score so I would expect you to see the missing peptides in the Library Explorer.
-- Nick