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
 
shulian13 responded:  2025-11-11 19:02
Dear Skyline team members,
       First, MSFragger-Glyco got stuck at this step: "[2025/10/29 12:15:49] Importing peptide lists from file peptide_list.txt...". For details, please refer to the last step of log file (log_2025-10-29_13-55-17 _lian shu). Second, I have uploaded the relevant files to the location you specified, named lian shu.zip. Regarding my search, I am not sure if anything is missing— such as a library or a software platform. Looking forward to your reply.
Lian
 
Nick Shulman responded:  2025-11-13 05:43
Thank you for uploading "lian shu.zip".
The file "fragpipe.sky" is a blank Skyline document with definitions for about 700 variable modifications.
I was hoping to use Skyline to add the file "fragpipe.blib" to that Skyline document and then use "View > Libraries > Library Explorer" and then "Add All" button to add all the peptides to the document.
Unfortunately, bringing up the Library Explorer takes hours. I think all of the modification definitions are causing Skyline to take an exponentially long amount of time to do certain things.
It is likely that this is also the cause of you getting stuck at the "Importing peptide lists" step.

It seems that there are some performance problems with Skyline and very large numbers of modifications which we will need to fix before this scenario will work for you.
-- Nick
 
Nick Shulman responded:  2025-11-13 06:54
Actually, there is a simple workaround which you can do to be able to use the document that fragpipe gave you with the library the you created.
If you go to the "Modifications" tab at "Settings > Peptide Settings" and change "Max variable mods" to "1" then things will not take too long.

I'm attaching a Skyline document where I changed Max variable mods to 1, and then added "fragpipe.blib" to the document and then went to "View > Libraries > Library Explorer" and pushed the "Add All" button.
This presumably added all of the peptides that had been found in the library to the document.
Is this helpful? Are these the modified peptides that you were hoping to analyze?
-- Nick