There are a couple of ways of inserting peptides into Skyline.
One way is with the "Edit > Insert > Peptides" menu item. There you would paste in a list of peptides sequences with one peptide per line.
Another way to get peptides into Skyline is with the "File > Import > Transition List" menu item.
In this case, the file can either be a tab-separated or a comma-separated file.
With a peptide transition list, one of the columns should be the peptide sequence, another column the precursor m/z and another column the product m/z.
When you import a transition list, Skyline will try to figure out which column is which. If Skyline is not sure, you will be presented with a form where you can tell Skyline how to interpret the columns.
With a peptide transition list, you only tell Skyline the precursor and product m/z's. You do not tell Skyline whether it is a y or b ion or the charge state. Skyline figures out the identity of the fragment ions from their product m/z's.
What people usually do is add peptides to the document using the "Edit > Insert > Peptides" menu item. Then, go to the "Library" tab at "Settings > Peptide Settings" and push the "Build" button. Tell Skyline that you want to use Koina to build the library. Skyline will create a library with predicted spectra for the peptides in the document and then Skyline will choose appropriate y and b ions based on the predicted spectra. You might have to go to the "Filter" and "Library" tabs at "Settings > Transition Settings" to get Skyline to choose the types of ions that you were hoping for.
When you insert peptides into the document using the "Edit > Insert > Peptides" menu item, those peptides will usually end up in one big list. If you want to organize the peptides by the protein that they belong to, you can use the "Refine > Associate Proteins" menu item and provide a FASTA file. If your peptides are non-tryptic, we recommend using Skyline-daily instead of Skyline 24.1 because we Associate Proteins does not work very well with non-tryptic peptides in Skyline 24.1.
-- Nick |