Query in DIA peak extraction and Groupwise comparison

Query in DIA peak extraction and Groupwise comparison Sangram  2023-02-28 23:54

Hi Skyline Team,

I am analyzing DIA data from for cell line proteome generated on Sciex TripleTOF 6500.

Nick and I have previously tried to understand the Isolation Scheme issue and now I do it by manually inputting it (and have it saved) as we use variable window. But I have noted that when I use only DIA data to generate a spectral library and quantify, Skyline produces only 1800 proteins while from the same files DIA-NN is producing 3500 proteins. But if we use DDA data to build spectral library, then we are able to generate 6500 proteins and quantify. We have also used it to create a groupwise comparison. Now my queries:

  1. We know that DIA produces more data then why are we able to capture much less protein than DDA. What can we check and improve ?
  2. In the groupwise comparison, the proteins of interest (we have RNAseq for the samples and looking for those genes) are all non-significantly expressed across groups. How can that be possible even the proteins of highly expressed genes ? How can we check that and also what method is used to calculate the fold change (need it for writing the methods).

P.S. I have the skyline zip [9Gb] in Onedrive and can share as per your convinience.

Much gratitude for all the help and advice.

Best regards

Nick Shulman responded:  2023-03-01 07:30
The following support request has a description of how the fold changes are calculated:

I am not sure I understand your other question. It sounds like you are saying that you imported DIA-NN peptide search results and the number of proteins in your Skyline document is not as much as DIA-NN says that you should have.
I would recommend that you first ignore the number of proteins that you have and instead only focus on the number of peptides that you have. Once you have all of the right peptides in your document you can use the menu item "Refine > Associate Proteins" to move those peptides around to different proteins and your choices about FASTA file and protein grouping will affect how many proteins you end up with. (In order to figure out how many peptides are in your Skyline document you should first use the menu item "Refine > Remove repeated peptides" and then look at the number of peptides displayed either in the status bar at the bottom of the Skyline window or by looking at the number of rows in the Document Grid when you are looking at the "Peptides" report).

One way to be more certain that you get all of the peptides from your search results into Skyline is to use the menu item:
View > Spectral Libraries
and then press the "Add All" button.

If you leave the "Associate proteins" button unchecked when you press the "Add All" button, then all of the peptides which had been missing from your document will get added to a Peptide List called "Library Peptides" and you can look at those peptides and figure out why they were not included in your original set of peptides that you got when you imported your peptide search results. The most common reasons have to do with the settings on the "Digestion", "Filter", "Library" and "Modifications" tabs of the "Settings > Peptide Settings" dialog, and the "Filter", "Library", "Instrument" and "Full Scan" tabs of the "Settings > Transition Settings" dialog.

You certainly can send us your data files and we can figure out what is going wrong. We usually learn a lot whenever we look at large datasets like this, and we are in the early stages of creating a process by which you will be able to create a Skyline document which has all of peptides from your DIA-NN search results.

I will send you an email.
-- Nick