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importing target and decoy libraries for crosslinked peptides

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importing target and decoy libraries for crosslinked peptides anzuki  2025-08-14 08:56
 

Dear Skyline-Team,

I would like to analyse crosslinked peptides for quantitation using a spectral library that already contains all the necessary information, including decoy fragments. I have tried this using the assay library import. Unfortunately, I failed to import the csv file because the peptide mass difference was too much from the calculated one by the sequence. That would be expected since crosslinked peptides are larger (two peptides is one sequence) and the automated calculation would naturally fail. But how can I fix this issue? Is there a way to still import my library? Maybe by converting the sequence to strings like that: GQKNSR-GQKNSR-[PhoX@3,3]

Or is there another way to do it?

Thank you very much for your help.

Best wishes,
Franze
 
 
Nick Shulman responded:  2025-08-14 09:17
Unfortunately, it appears that you cannot use the crosslinked peptide sequence syntax "GQKNSR-GQKNSR-[PhoX@3,3]" in a transition list.

You might be the first person to try creating a transition list for crosslinked peptide sequence.
The usual thing that people have been doing is creating a BiblioSpec spectral library from crosslinked peptide search results.
The first thing that you need to do is create a .proxl file from your crosslinked peptide search results.

Do you have crosslinked peptide search results? What format are they in?
You can find a list of supported formats here:
https://proxl-web-app.readthedocs.io/en/latest/using/upload_data.html

Let us know if your format is not in the list because we might be able to create a proxl converter for whatever you have.

By the way, what is the chemical formula of the PhoX crosslinker?
-- Nick
 
anzuki responded:  2025-08-15 06:38
Hi Nick,

I have tried to use the proxl converter but both software tools I am using are not supported: MS Annika with Proteome Discoverer and xiSearch/xiFDR. Therefore, I went to the assay library option.

The monoisotopic mass of the PhoX crosslinker is 209.9718097 after the crosslink reaction with the composition C8H3O5P. This also includes the reaction to both peptides of the crosslinker.

Best wishes,
Franze
 
anzuki responded:  2025-09-02 06:59
Hi Nick,

do you think it would be possible to create a proxl converter to convert my Library? Or maybe import my library differently, then what I am trying at the moment?

Any recommendation would be helpful.

Best wishes,
Fränze