Defining Isolation Schemes for Bruker timsTOF DIA data Premy  2023-01-13 10:01

Thanks again for all the support with Skyline!

I am developing a DIA-IM Lipidomics pipeline on Skyline and was wondering whether an isolation scheme would need to be defined for Bruker timsTOF data or if the isolation scheme can be read from my raw files using "Results Only"?



Nick Shulman responded:  2023-01-13 11:29
I am not sure, but I believe that Skyline can read the isolation windows from timsTOF data.

One way to check this is to use the "Import" button in the Edit Isolation Scheme dialog. If Skyline is able to read the isolation scheme via that Import button then you could have left the setting as "Results Only".

If you are using the "File > Import > Peptide Search" wizard to do a DIA Umpire search then I think you do need to have pre-specified the isolation windows. I do not know the details on whether that is always necessary, or whether it was just for the data that I happened to be looking at.
-- Nick
Premy responded:  2023-01-13 11:41
Hi Nick,

I checked with the "Import" button in the Edit Isolation Scheme dialog and it was able to read the isolation schemes, thank you!

I am doing lipid analyses using the Molecule interface, so I don't think the search wizard is available? I am currently analyzing the raw files using a transition list from LipidCreator, a developed IM library and iRTs.

Appreciate all the help!

Brendan MacLean responded:  2023-01-18 11:52
True. Import Peptide Search is only available for peptides. However, pre-specifying the isolation list as Nick has described also allows Skyline to enforce that your chosen precursors are measured by it. There is also a setting that allows you to prohibit use of fragment ion m/z values within the precursor isolation range, because that range can be noisy. Though, I am not sure how well-supported that is for small molecules.

Those are the only 2 cases I can think of where pre-specifying the isolation range provides extra functionality. Well, there is also the ability to visualize the isolation scheme with the "Graph" button after you use "Import" as you said you have. This visualization can help you confirm that the isolation scheme is what you think and has no issues, which could easily get past you with "Results Only" and cause much more confusing outcomes if there are actually unexpected issues in the isolation scheme.

Thanks for your post to the Skyline support board.

Premy responded:  2023-02-23 15:14
Hi Brendan and Nick,

Thanks for the help!

I had a follow-up question that just came up. Is it possible that some schemes cannot be identified from "results only"? I was manually checking through "Import Results" in the Edit Isolation Scheme dialog to see if my different window schemes could be read, and a few came up with an error as follows: "Failure reading isolation scheme, no repeating scheme found in data". These were both variable window schemes (although other variable window schemes seem to be okay when read).

Looking at the data itself in Skyline, the peaks do have similar properties to what I would expect the data to look like for these cases that had a failure.

Thanks again,