Identifying a significant protein in Skyline but it's not found in raw files. renad almahdali  2020-10-15

Dear Skyline team,

First, thank you loads for this amazing efficient software!
I am now working on constructing human proteomics profile from PBMC samples using Sciex TripleTOF 5600 and DDA method. I happened to come across a significant (highly expressed) protein in Skyline but when I wanted to check it in the proteins list exported from ProteinPilot raw file I didn't find it. My question is, is it possible to find a protein in Skyline software only but not from the original files?? I even tried to look it up by its protein name and accession including it synonyms.

Waiting for your support guys,
Renad Abdulrahman

Nick Shulman responded:  2020-10-15
I am not sure I understand your question. You are saying that there is a protein in your Skyline document that was not present in your peptide search results, and you are wondering how it got there?

 When you do "Import > Peptide Search", the last step of the wizard asks you to provide a FASTA file. Skyline performs an in-silico digestion of the protein sequences in that FASTA file, and you will get all of the peptides added to your document which match your peptide filter settings. Usually, this means that you will only end up with peptides which were in your peptide search results, but there are a few ways I think that you could end up with peptides (and therefore proteins) in your document that were not found in those search results:
1. If your document had already had another library before you started the Import Peptide Search wizard.
2. If the "Pick peptides matching" setting at "Settings > Peptide Settings > Library" was something that said to include things that are not in the library: "Filter" or "Library or Filter".

I might not have understood your question at all. If you would like, you can send us your Skyline document and we can take a look.

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.
Otherwise, you can upload it here:
-- Nick