These are what we are currently recommending for settings on the Q-Exactive series with our software.
https://bitbucket.org/searleb/encyclopedia/downloads/dia methods setup.pdf

The date would get demultiplexed by msconvert and the results imported into Skyline.

Thank you, I was referring to Table 2, column b, of this document which has variable width windows. To clarify, in Figure 6, panel b, would the isolation window setting still be 24 m/z?

If you are using a variable width you would have a different width for each window. We don't use variable width windows. We use a fixed width but overlapping window. The overlapping window enables us to demultiplex the window to 50% of the isolation width. However that requires using Proteowizard msconvert to demultiplex the file first.

Cheers,
Mike

Is your method a single injection? Would you need the demultiplex step if you split the overlapping window lists into two separate runs?
Do you have a protocol available for the demultiplex step?

Thanks again!
Nicole

Hi Nicole,
Here also is some explanation from Ludovic Gillet on setting up DIA methods for Q-Exactive and Lumos instruments in his presentation from this summer's DIA/SWATH Course at ETH (starting at about minute 41 of the presentation):

https://youtu.be/iDdEF0GKfXo

It looks to me like the method setups he shows allow individual control of isolation window widths. Because, as Mike says, the MacCoss Lab uses fixed width overlapping windows with post-acquisition demultiplexing (using ProteoWizard MSConvert) the methods can be generated with a simple inclusion list and a single fixed isolation width for all windows. But, it is certainly also possible to set up variable width isolation on these instruments and I think the method setup Ludovic explains should show you how.

I have also asked Ludovic himself to comment on this thread, but it being the weekend, we may not hear from him until next week.

Thanks for posting to the Skyline support board and initiating this discussion.

--Brendan

To follow up on your question, we perform all of our quantitative analysis using a single injection. In the PDF I sent, columns d and e of table 2 are concatenated. You get a single concatentated list from Skyline and you can see as example of it pasted into the inclusion list in Figure 7.

If the windows are overlapped like described in the pdf, we demultiplex each 24 m/z window to 12 m/z using linear algebra. We are able to take data collected with 24 m/z windows and demultiplex them down to the equivalent of collecting 12 m/z because of the overlap. This can be done using the latest version of msconvert.exe from http://proteowizard.sourceforge.net/. Attached is a screenshot showing the suggested parameters for doing this.

You can also do this from the command-line using:
msconvert.exe --zlib --64 --mzML --filter "peakPicking true 1-" --filter "demultiplex optimization=overlap_only" --simAsSpectra *.raw

I will point out that the Fusion Lumos method editor is quite a bit different than the Q-Exactive method editor. We are working on better documentation for this.

-Mike

Thank you both! I will give it a try.

Here also are a couple more PDFs, one from Ludovic (the slides he presents in the video) and one from Roland Bruderer at Biognosys.

Perfect! Thanks again for your help Brendan.

Hi Mike,
I attempted to use your overlapping window method with the demultiplexing step... and it failed - here is the error message from MSConvert:
Failed - System.Exception: SpectrumToIndices() Number of demultiplexing windows changed. Window boundary tolerance may be set too low.
at pwiz.CLI.msdata.MSDataFile.write(MSData msd, String filename, WriteConfig config, IterationListenerRegistry iterationListenerRegistry)
at MSConvertGUI.MainLogic.processFile(String filename, Config config, ReaderList readers)
at MSConvertGUI.MainLogic.Go(Config config)

Any suggestions?

Thanks,
Nicole

Hi Nicole,

Are you able to provide us with the raw file that gave that error so we can debug it on our end? You should be able to upload it to our share site here: https://panoramaweb.org/project/MacCoss/atkeller/Transfer/begin.view?
If not, I may still be able to provide some guidance.

Thanks,
Austin

I can provide the raw file, however the link states that I don't have permission to.
Is there another way to get you the file?

Hi Nicole,

Sorry about that. You should be able to upload the file here instead: http://skyline.ms/files.url