Can you send us your Skyline document?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries.

If that .zip file is less than 50MB you can attach it to this support request. Larger files can always be uploaded here:
https://skyline.ms/files.url

When you are building a spectral library from peptide search results, Skyline does not need to know what the modifications are because what gets stored in the .blib file is just the mass of the modification. However, when you want to do something with the library, such as browsing it using "View > SpectralLibraries", Skyline tries to figure out what the names of the modifications are which correspond to the modification masses in the library. If all of the modifications that you are using are standard Unimod modifications then Skyline usually does a good job of guessing which modification mass is which. It sounds like you are using nonstandard modifications, in which case you should go to "Settings > Peptide Settings > Modifications" and tell Skyline about all of the modifications that you are using.

After we see your Skyline document we will probably have a better understanding of what is going on.
-- Nick

Hi Nick,

Sorry for the delay. I've uploaded the .zip file to the link you provided. It's NK_FBN1_D1031A.sky.zip.

Thanks,
Nick Kegley

Thank you for uploading that Skyline document.
When I go to:
View > Spectral Libraries
Skyline shows me the attached "Add Modifications" dialog with the list of modifications that are in the library but which have not been declared in the "Settings > Peptide Settings > Modifications" page.
The list box at the top contains modifications which have the same mass as a unimod modification. If the unimod modification is the correct definition for a particular modification then you can check the checkbox next to the unimod modification name and Skyline will use that as the definition while showing you the Spectral Library Explorer.
When you are looking at the Spectral Library Explorer, peptide sequences with unexplained modifications will be missing the peptide icon next to them. You won't be able to add those peptides to your document using the "Add" or "Add All" buttons in the Spectral Library Explorer.

If you check all the checkboxes next to the unimod modifications in the "Add Modifications" dialog, then more peptide sequences in the Spectral Library will have the peptide icon next to them.
If you add a peptide to your document which is using one of the modifications that you had checked in the Add Modifications dialog, then the unimod modification will be added to the list of modifications in the "Settings > Peptide Settings > Modifications" page.

In the "Add Modifications" dialog, the list at the bottom contains modifications (e.g. "T[+324.10565]") that do not the same mass as any unimod modifications. For each of the masses in that list you will need to go to:
Settings > Peptide Settings > Modifications
and using the "Edit List" button next to the "Structural Modifications" list box to add a modification definition.

Does this answer your questions? Are you getting stuck somewhere?
-- Nick