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

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

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

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

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

Now that the method is functional. We expect that Skyline (via View > Libraries > Library Match) be configured to report key m/z values—specifically for the oxonium ion from N-glycans (e.g., 138.06, 204.09, 274.09, 292.1, 366.14, and 657.24; integer values are sufficient), B ions, and Y ions—along with the scan numbers for identified glycopeptides. This information is essential for rigorous manual validation and confident spectral assignment. Although presenting the full suite of fragment ions (B and Y) remains technically challenging, reporting the oxonium ion m/z values alongside their corresponding glycopeptide scan numbers is a readily achievable objective.
Thank you very much！

Lian
Nov,26,2025

Hey Lian,

Attached are a subset of slides that were presented in Skyline Online 2025 where I show how some glycan-specific ions can be defined in Skyline.

Oxonium ions and N-glycan core Y-ions can be defined in a semi-straightforward way.

I hope the slides helps in clarifying it, otherwise let me know if you have more questions.

Sincerely,
Juan C.

Hi Juan,
Thank you so much for your prompt response and for sharing the subset of slides from Skyline Online 2025! They are incredibly helpful in clarifying how to define glycan-specific ions—especially oxonium ions and Y-ions—in Skyline. The step-by-step explanation in the slides makes the semi-straightforward approach much easier to follow, and it may directly addresses the questions I had.
I’ve gone through the slides briefly and they provide clear guidance for my current work.I will attempt to apply the methods you shared in my work. If I encounter any further questions while applying these methods in practice, I’ll feel free to reach out.
Again, I really appreciate your time and support. Have a great day!

 Lian Shu
Nov,26,2025