I would suggest beginning with an existing Skyline document that already works for you, with the idea that it has peptide digestion settings that you like. Delete everything in the Targets tree, then drop the FASTA in and see if that doesn't work.

Failing that, you will have to think about the various Peptide Settings that control how Skyline predicts protein digestion.

Good luck, and thanks for using the Skyline support board.

Brian

It seems likely that you have chosen a spectral library in your Peptide Settings - Library tab which does not contain any spectra from this protein. Otherwise, as Brian, suggests some other setting is causing Skyline to exclude all of the peptides from this protein. If you change the peptide selection from "Library" to "Filter", you should see more peptides, though they may be less useful than you had hoped if they have no associate library spectra.

You can also hover your mouse cursor over the protein name that ends up in your Targets view and then click on the drop-arrow-head that shows up to the right of the name to bring up the pick list for peptides. Then click on the button on the right with the funnel icon. This should show you all the peptides Skyline knows about that fit within your instrument measurement range, and I expect you will see that they do not have associated library spectra.

Or you may also have a better library for this target.

Good luck. Thanks for posting the screenshot images.

--Brendan