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Generating CE-Optimization for timsTOF using Targetlist (without real data) does not work

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Generating CE-Optimization for timsTOF using Targetlist (without real data) does not work gianin thomann  2025-02-17 05:32
 

Dear Skyline team,

I generated a transition list with peptides from from DIA-NN (report and speclib) data to go further into prm-PASEF, i.e. optimization of CE (see Skyline-Targetlist-test.csv). However, the isolation list export for Bruker timsTOF fails due to missing ion mobility values:

Skyline

All targets must have an ion mobility value. These can be set explicitly or contained in an ion mobility library or spectral library. The following ion mobility values are missing:

AEATTLHVAPQGTAMAVSTFR+++
DADPDTFFAK++
DSEITFIKK++
EFNAETFTFHADIC[+57.021464]TLSEKER+++
FKN[+0.984016]GMLHGDK++
FVC[+57.021464]NSGYK++
IPAC[+57.021464]IAGER++
ITQVLHFTK++
LFLEPTRK++
MDVFSQNMFC[+57.021464]AGHPSLK++

OK

But, the values are given in the transition list during the import as Explicit Ion Mobility which seem to be successfull as the corresponding value is given in the spectral library view afterwards. So at least, the value is somewhere, but not accessible for the export...
Note, the box "Use spectral library ion mobility values when present" (Settings > Transition Settings > Ion Mobility) is checked.

Do you have any advice?

Best,

Gianin
 
 
Nick Shulman responded:  2025-02-19 05:52
Gianin,

Can you send us a .sky.zip file instead of the .sky file that you attached?

In Skyline you can use the menu item:
File > Share
to create a .sky.zip file containing your Skyline document and supporting files including spectral libraries.

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

The .sky file that was attached to this support request did not have any peptides in it. I might have been able to import the transition list that you attached, but I am not sure exactly which options you chose in that process, so it would be best if you could send us a .sky.zip with all of your peptides already in the document.
-- Nick
 
gianin thomann responded:  2025-02-19 07:47
Dear Nick,

Sure. Please find attached the zip.

Meanwhile I managed to create a imdbs using real data, then it seems to work to export an Isolation List by using the created library (not shown in the Skyline_test file). But still it is a bit confusing that it does not work via the spectral library although checked in the settings...

Using the created values however, the exported Isolation list for CE-optimization (Bruker timsTOF, Single Method, CE Optimization, Method Type Standard) exports CE of 1-5 eV for every target, even if I create it based on the real prm-PASEF data (where the real CE of each transition should be accessible for skyline). Do you know if the CE optimization is still limited in case of timsTOF data?


Gianin
 
Nick Shulman responded:  2025-02-20 16:04
Thank you for sending that Skyline document.
It looks like the reason that Skyline is telling you that targets do not have an ion mobility value is that Skyline does not know what ion mobility window width to use.
If you go to the "Ion Mobility" tab at "Settings > Transition Settings" and choose something for "Window Type" (e.g. "Fixed") and specify a non-zero window width then Skyline would be willing to export a timsTOF isolation list for you.
-- Nick