Hi Si,
Do you have any sort of FASTA format file which was used as input into Maxquant? I am not sure I understand how a peptide search engine would function without a FASTA file that lists the amino acid sequences of the proteins to consider. Just import that FASTA file where the tutorial uses Fasta.txt. Fasta.txt is just an example of a FASTA format file. You can use an FASTA format text that overlaps with the peptides of interest.

This is your easiest path forward. Hope it helps.

Thanks for using Skyline in your research.

--Brendan

Hi Brendan,

Thanks for the reply. It worked!
Now I'm having a new issue when associating proteins to the peptides. After I checked 'Associate Proteins' and clicked on 'Add all', a peptide filter window showing that ~10,000 peptide not matching any proteins (See attached), which ended up with on ~800 total proteins instead of ~1500 proteins from the search result. I had my peptide setting window shots attached (Capture 2-6). I don't know if that would be the reason to cause the loss of proteins. Or is there any other settings that I need to change? Thanks a lot!

Si

Hi Si,
Sorry for missing this follow-up question for so long. It is hard for me to say what is going on without seeing the list of peptides that end up without a protein association. I think the best course of action in a case like this is to tell Skyline you want to add those peptides anyway, and then have a look yourself at what it adds without a protein association, and then search for them in the FASTA file you used to create your background proteome.

If you can't find them in the FASTA file, then you have your answer on why Skyline can't either. If you can find them, then the most likely cause is that the peptides are not fully trypticly cleaved. I do see in the Spectral Library Explorer window that you sent at least one such peptide with a C-terminal Valine. Harder to tell whether something might not be the result of tryptic cleavage on the N-terminus, but that would have the same impact.

It looks to me like you may have used semi-cleavage or unconstrained cleavage in your search, and that has allowed some peptides which are not fully tryptically cleaved into your library.

If you actually want to include this in your targeted analysis, the only option in Skyline, currently, is to put them into peptide lists. Skyline doesn't yet support making a protein association for peptides not fully cleaved.

If you don't want these peptides included in your targeted analysis, you can just choose not to add them in the form you have shown, or you can use the method of importing the FASTA file into your document, rather than using the Spectral Library Explorer. Then Skyline will include only the subset of peptides from your library that match your current settings and appear in your FASTA file.

Hope this helps. Thanks for posting your support request, and again sorry for not noticing you had made a follow-up request until now. Mid-September was very busy with a week-long course in Barcelona.

--Brendan