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Visualising PRM data, knowing the targets of interest, but without an experimental spectral library

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Visualising PRM data, knowing the targets of interest, but without an experimental spectral library chloe baldreki  2026-01-27 08:21
 

Hi Skyline team,

I'm getting myself a little confused about how to set up a document from scratch for some PRM data, for which I have a list of target peptides, but no experimental spectral library. I went through the process of setting up the peptide settings, including the use of PROSIT in the library tab (as I hoped this could be used instead of an experimental library), and then the transition settings. I then inserted my list of peptides into the document (which matched to my protein of interest from the background proteome), however the document did not give me any precursors or transitions. I therefore could not import my data for visualisation. Have I missed something essential here?

Relatedly, I also saw that is was possible to 'Run Peptide Search' using MSFragger, which I thought might be a way around this but couldn't find a tutorial related to this, and unfortunately the search I set up gave me an error message. Is this a viable approach?

Do you have any guidance on how to visualise my PRM data, knowing the targets of interest, but without an experimental spectral library?

Many thanks in advance for your help!
Chloe
 
 
Nick Shulman responded:  2026-01-27 08:43
You must insert the peptides into your document before you use the Prosit feature in Skyline to create a spectral library.
The library that you create will have predicted spectra for the peptides in your document. It sounds like you created the library when you did not have any peptides in the document, so you ended up with an empty spectral library. Then, when you did insert your peptides, Skyline saw that the spectral library did not contain spectra for these peptides and therefore did not give you any precursors or transitions.

Yes, you should be able to run an MSFragger search in Skyline. What error message did you see?

Note that if you have Bruker timsTOF data, then you must do the MSFragger outside of Skyline and import the results using File > Import > Peptide Search. The reason for this is that Skyline only knows how to run MSFragger version 4.1, and you must use at least version 4.2 of MSFragger with timsTOF data because otherwise the spectrum identifying numbers cannot be understood by Skyline.
-- Nick
 
chloe baldreki responded:  2026-01-27 08:55
Hi Nick,

Thanks very much for your fast and helpful response!

I will try setting up my document again making sure to add my peptide list before setting PROSIT up. Hopefully this will resolve my issue.

Unfortunately I clicked off the message without noting its cause, but I am using timsTOF data, so possibly it related to this. I will also try searching the data with FragPipe and importing that way to see if this provides anything different.

Many thanks!