Replicate Comparison: Replicate Data not Showing up noaheearls  2022-08-31
 
I am trying to analyze a dataset with 9 replicates. The Replicate Comparison for the Retention Times and Peak areas only present values for one replicate, though the chromatography shows peaks for the other replicate. Is there a setting or method to attribute the data in the replicates in a way that can be viewed in the replicate comparison? See Attahched for an example peptide:
 
 
Nick Shulman responded:  2022-08-31
I see that Skyline is showing you the Total Ion Current normalized values in the Peak Area graph.
If you want to see the unnormalized values you can right-click on the Peak Area graph and choose something else for "Normalized To >".

It might be that the reason that the values are blank for most of the replicates is that Skyline does not have a value for the Total Ion Current Area.
You can use the Document Grid to look at the Total Ion Current Area values for all of the result files.
If you would like to learn about the document grid you should look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

There are many reasons that Skyline might not have a Total Ion Current for a replicate. If you send me your Skyline document and raw files, I could figure out what happened.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
If that .zip file and your raw files are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url

-- Nick
 
noaheearls responded:  2022-09-01
I shared the files in the larger upload location under the name "SkylineSupport.zip". Let me know if you need anything more from me!

Thanks!

Noah
 
Nick Shulman responded:  2022-09-01
Thank you for sending your Skyline document and raw files.
The DIA isolation scheme that you have specified at "Settings > Transition Settings > Full Scan" does not match what is in your raw files.
Your isolation scheme has windows which are 400-500, 500-600, etc.
The isolation scheme in your raw files have overlapping windows 400-408, 404-412, etc.

I see that you extracted chromatograms from mzML files, so I imagine that your mzML files had deconvoluted windows in them.
You can use the isolation scheme called "Results Only". This isolation scheme tells Skyline to trust the isolation windows that appear in the raw file. There are cases where you need to tell Skyline ahead of time what the isolation windows are going to be, such as when you are doing a DIA Umpire peptide search, or there are certain types of mass spectrometers where Skyline does not know how to read the isolation window, but, in general, for Thermo files, you can use the isolation scheme "Results Only".

I expect that this incorrect isolation scheme would seriously confuse Skyline, but I am not sure why that would result in the exact behavior you are seeing.
I would recommend that you change the isolation scheme to "Results Only" and then go to:
Edit > Manage Results
and tell Skyline to Reimport all the files.
-- Nick