Skyline 23.1 has an import wizard to let you easily run a library-free EncyclopeDIA search on your gas-phase fractionated (narrow window) and single injection (wide window) DIA results. You choose a FASTA and Skyline generates a Prosit library from it and uses that to seed the EncyclopeDIA search. After the search, Skyline automatically imports the chromatogram and quantification libraries that were created.

1. Start the wizard

2. Choose FASTA

• Enzyme and missed cleavages determine which peptides will be predicted
• Prosit prediction on a whole proteome can take a while so for a quick test choose a targeted FASTA

3. Pick Prosit settings

• m/z min/max can be set to filter peptide predictions on m/z
• Precursor charges between 1 and 6
• Default NCE is the NCE at the default charge

4. Pick GPF (narrow window) DIA results

• EncyclopeDIA searches these to build chromatogram library

5. Pick single injection (wide window) DIA results

• EncyclopeDIA searches these to build quantification library

6. Pick EncyclopeDIA settings

• Wizard page currently only exposes peak tolerances
• All EncyclopeDIA settings can be edited in “Additional Settings”

7. Watch progress

8. After search finishes, import proceeds with the import peptide search wizard where the EncyclopeDIA libraries have been preselected