Import Peptide Search - No spectra in library

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Import Peptide Search - No spectra in library claire tonry  2021-09-15
 

Hi, I am trying to import peptide search data into Skyline for dia analysis. The library was generated from an 'open search' of dda files (timsTOF) that were converted to mzML. When I try to use the individual .pepXML files (15,000 - 20,000 KB) for each sample or the 'interact.pep' file (177,609 KB) Skyline tells me that it cannot find any spectra for the library. I also tried using the library.tsv.speclib file generated but this also caused an error as 'has version 3 bu BiblioSpec only supports up to version 2'. These dda and associated dia files were previously searched in Spectronaut software with no issues. Would you have any ideas as to why Skyline cannot read any spectra from the output files generated from the fragpipe search? The objective of this search is to identify PTMS which is why I wanted to use output from a fragpipe open search

 
 
Nick Shulman responded:  2021-09-15
Another user reported a problem with Fragpipe which was happening because BiblioSpec was trying to find the spectra in the .d raw file folder, but the spectrum identifiers in the pepxml only made sense if you were to look for those spectra in the .mgf file.
Here's the support request for that:
https://skyline.ms/announcements/home/support/thread.view?rowId=52421

I am not sure whether that might be the same problem that you are running into. If it is, the workaround is to make sure to delete the .d folder, and any .mzML file that you might have in the same folder as your pepxml file, so the only thing that BiblioSpec is able to find when it goes looking for spectra is going to be the mgf file.

This other user described the problem as a hang because it was taking a very very long time to not find the spectra in the file it was looking through. However, I could imagine the behavior could also manifest itself as no spectra found.

If that does not fix your problem, then you should send us your files and we can try to reproduce the problem.
You can zip up your files and upload them here:
https://skyline.ms/files.url

-- Nick