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Offset windows in DIA analysis sweaver4  2023-07-31 07:44
 

Hello,

I'm looking through some DIA bottom-up proteomics data acquired on a timsTOF where the isolation windows were 24 m/z, but offset (i.e. 400, 424, 448... 412, 436...). How should I setup the transition settings to reflect this? I can point the isolation scheme to the .d file which successfully pulls in the m/z values used for the windows. Should I set the Deconvolution to overlap in this window?

My goal is to look at the chromatograms and have the intensity traces for each fragment ion reflect only MS2 signal that was present in spectra where the precursor isolation windows were appropriate. (So if an example precursor ion was 430 m/z, fragments would show up in isolation windows 424-448 and 412-436, but not 400-424). Is this how the chromatogram extraction is automatically designed, or do I have to perform additional deconvolution before uploading my .d files?

Thanks very much for any help, and let me know if I can clarify anything! I looked through the tutorials and documentation and couldn't find any specific examples for this, but apologies if it is somewhere and I missed it.

Simon Weaver.
 
 
Nick Shulman responded:  2023-07-31 10:30
We recommend that you use ProteoWizard MSConvert to create .mzML files with demultiplexed spectra and tell Skyline to extract chromatograms from those .mzML files.

In the MSConvert graphical user interface, you can add a filter called "Demultiplex". This works much better than the thing that we implemented in Skyline.

You should not use the "Deconvolution" dropdown in the "Edit Isolation Scheme" window in Skyline. That feature was implemented in Skyline before we actually understood how to do this correctly.
-- Nick
 
Mike MacCoss responded:  2023-07-31 10:47
We've never tested the demultiplexing on timsTOF data before. We would like to get input back on how this works. We have a Tip on how to generate an isolation list that we believe should work.
https://skyline.ms/wiki/home/software/Skyline/page.view?name=OverlappingWindowScheme

We also have a tip on how to perform the demultiplexing from Thermo RAW files.
https://skyline.ms/wiki/home/software/Skyline/page.view?name=DemultiplexingOverlappingDIAWindows

I don't think there is any reason why the demultiplexing shouldn't work with timsTOF data but we have never tried it. Please let us know how this works.

Cheers,
Mike
 
sweaver4 responded:  2023-07-31 11:03
Sounds good, I will try to demultiplex using MSConvert, and I can respond here with how it worked once I get a chance to look at the data. Thanks for the help!
-Simon