• Sign In
  1. Requests

Exploris DIA - setting up isolation scheme in Skyline from file

support

View Request

view list print
Exploris DIA - setting up isolation scheme in Skyline from file caitriona scaife  2025-01-13 07:41
 

Hi,

I have run two samples on our Exploris 480, one sample in DDA and the second using DIA (selecting the Thermo DIA template from their Method Editor). I am now having an issue when trying to set up the DIA Isolation Scheme in Skyline.

Previously for diaPASEF files I had been able to set up the DIA isolation scheme in Skyline by selecting the "Prespecified isolation windows" button and using the "Import" link to locate and extract the window information directly from the file. However when I try this with the Thermo DIA file I get the following warning (depending on whether or not "Specify Margin" has been selected).

If I select the "Specify margin" option I get "There are gaps in a single cycle of your extraction windows. Are you sure you want to continue?" The Thermo method does have a Window Overlap of 1 m/z and the margin in Skyline is showing up as 0.5002.

If I de-select the option I get "There are overlaps in a single cycle of your extraction window. Are you sure you want to continue?"

I would appreciate if you could help figure out what I am doing wrong. By the way I have gone ahead and used both versions of the Isolation schemes, with and without margins to extract the targets and targets are extracted in both cases.

Thanks in advance
Caitriona
 
 
Nick Shulman responded:  2025-01-13 07:54
Can you send us one of your .raw files?
Files which are less than 50MB can be attached to these support requests. Larger files can always be uploaded here:
https://skyline.ms/files.url

-- Nick
 
caitriona scaife responded:  2025-01-13 09:43
Zipped file uploaded
 
Nick Shulman responded:  2025-01-13 13:29
Caitriona,

It looks like you uploaded the DDA .raw file.
The "Import" button for isolation schemes would only be expected to work with DIA data. It tries to figure out the pattern of isolation windows that are being cycled through.
With DDA data, the mass spectrometer is deciding what to fragment based on what it sees, so there is no repeating pattern which makes it inappropriate for extracting MS2 chromatograms.

Can you upload your DIA raw file?
-- Nick
 
caitriona scaife responded:  2025-01-13 14:15
Sorry, DIA file should be uploaded now,

Caitriona
 
Nick Shulman responded:  2025-01-13 14:35
I went to the "Full Scan" tab at "Settings > Transition Settings". I chose "DIA" as the "Acquisition Method" and then I chose "<Add...>" from the Isolation Scheme dropdown.
In the "Edit Isolation Scheme" dialog, I clicked on "Prespecified isolation windows" and I pushed the "Import" button to read the isolation scheme from that .raw file.
Skyline was perfectly happy doing that as shown in the attached screenshot.

When you use the Import button to tell Skyline to infer the isolation scheme from the spectra that it finds in a .raw file, you do not need to worry about whether the "Specify Margin" checkbox is checked. Skyline will figure out whether there is a margin based on how the isolation windows overlap with each other.

It sounds like something is going wrong for you, but I am not sure what would be going wrong.
Can you post a screenshot of what you are seeing?
It might also help if you could send us your Skyline document.

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.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick
 
caitriona scaife responded:  2025-01-14 04:06
Hi Nick,

Yes, Skyline does read the isolation scheme ok however it is when I try to proceed to Configure Full Scan Settings that it issues the warning (attached screen snaps). I have tried to setup the Isolation scheme on a separate computer and even used an old version of Skyline Daily and am getting the same message each time - so could be "operator error"...

I have uploaded/shared a version of the Skyline document that was created using the Import DIA peptide search Wizard (EXPLORIS_TEMP). I just used the default settings presented in the Wizard.

(I apologise in advance if this turns out to be a stupid error on my part.)

Caitriona
 
Nick Shulman responded:  2025-01-14 10:02
You can safely ignore the warning "There are gaps in a single cycle of your extraction windows. Are you sure you want to continue?"

Skyline 24.1 considers that even a gap as small as 0.0001 m/z is enough to trigger that warning.
In Skyline-daily you will not see that warning because the gap has to be at least 0.001 m/z before you see that warning.

In Skyline 24.1, it is very difficult to look at numbers displayed in the Edit Isolation Scheme dialog and figure out which particular isolation windows Skyline believes have a gap greater than 0.0001 m/z. One reason for this is that even though Skyline is displaying numbers which have been rounded off to 4 decimal places, the number that Skyline is remembering has more decimal digits than that.

The first gap that Skyline notices that is greater than 0.0001 is between 339.904299 and 339.904407.
-- Nick
 
caitriona scaife responded:  2025-01-14 10:36
That’s good news 😀 and thank you for resolving the issue so fast,

Caitriona