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Strategy for establishing a PRM method for intact glycopeptides using Skyline?

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Strategy for establishing a PRM method for intact glycopeptides using Skyline? shulian13  2025-11-06 07:31
 

Since Skyline is not yet suitable for the identification and quantification of intact glycopeptides. I plan to use MSFragger-Glyco to search glycoproteomic data, simultaneously export a Skyline document, and utilize Skyline to construct a PRM method containing precursor ion and product ion information based on the Skyline document . Next, I will apply this PRM technology for targeted quantification of glycopeptides. However, I have encountered some difficulties: MSFragger-Glyco search was successful, but the Skyline document failed to export. Could you please recommend a strategy for establishing a PRM method for intact glycopeptides using Skyline or relevant team members to help me resolve this issue?
Thank you very much! I’m looking forward to your reply.
Best regards,
Lian Shu
 
 
Nick Shulman responded:  2025-11-06 17:31
Did MSFragger give you any sort of error message when it failed to create a Skyline document for you? This sounds like the sort of bug we would need to tell the MSFragger team about in order for it to get fixed.

My understanding is that MSFragger creates a Skyline document with very complicated modifications and neutral losses. You could do all that work yourself but it might be too complicated to do by hand.

If you send us your files and tell us what you did with MSFragger that failed to produce a Skyline document we can certainly investigate it and figure out who to send it on to.
You can upload files here:
https://skyline.ms/files.url

--Nick
 
shulian13 responded:  2025-11-06 19:30
Hi, Nick,
     I confirmed that the MSFragger-Glyco searching part was completed successfully. However, the Skyline document containing both precursor ions and product ions was not generated. A peptide_list.txt file was produced, which contains information on precursor ions but lacks product ion and neutral loss.
     I have uploaded the logs retrieved using MSFragger-Glyco and the generated Skyline-related documents to the shared archive. The last statement the software paused on:Importing peptide lists from file peptide_list.txt...
     Additionally, could you please share a tutorial on manually extracting parent and daughter ions, and neutral loss from Skyline?
    Thank you very much!

Lian