What difference in behavior are you seeing between different versions of Skyline?
Can you send us the exported transition list that you got with an older version of Skyline and the transition list that you are getting now?
Also, which older version of Skyline are you comparing this to?

I know that back in May we made a change so that if the slope of the equation was zero, Skyline would assume that it was an equation for the normalized collision energy (NCE) instead of being for a regular actual collision energy, so this change would affect Skyline 25.1 and later.

I am not sure what difference this NCE assumption would make, if any.
I am not seeing a difference in the exported transition list when I use Skyline 24.1 versus Skyline 25.1, so I might not have chosen the correct options on the Export dialog to manifest the problem that you are seeing.
-- Nick

Hi Nick,

I'm based in Australia so I can't access the old TeamCity archives of older versions but this assay was developed in 2023, and then recently worked on and saved in 2025 [srm_settings format_version="24.11" software_version="Skyline-daily (64-bit) 25.0.9.97 (ae4c99798)"]. The custom CE equation I'm using has the intercept at 50 and has 4 steps on each side, so 10,20,30,40,50,60,70,80,90 CE when interpreting the CE optimisation performed. But it gives 908.3, 909.3,910.3, etc.

Funnily enough, when I export a transition list in the optimising CE format, the CE values are correct, but features each step. It's only when I have "Use optimization values when present" checked, and Optimize by "Transition" that the exported optimized CE values are incorrect (e.g. 908.3 instead of 20)

Even more curiously, a similar approach with a different document [srm_settings format_version="23.1" software_version="Skyline-daily (64-bit) 23.1.1.268 (c58f1f56e)] using chemical formulae instead of precursor m/z, gives the right optimized CE values when exporting those optimized values into a transition list.

Cheers,
Chris

The way to run older versions of Skyline is to go to the "unplugged" installer page:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=install-64-disconnected

After you click "I agree" a link that says "Archive" will appear and you can see .zip files for all older released versions of Skyline.
After you download the .zip file you should unzip it (if you use Windows Explorer to do the unzipping then you need to "unblock" it before you unzip it, but if you use 7-zip to do the unzipping you won't have to have done that step).
Once it has been unzipped you can go find "Skyline.exe" in one of the subfolders and run it directly. There is also a "setup.exe" which you would use if you wanted Skyline added to your start menu but you probably don't want that if you're just verifying the behavior of an old version.

-- Nick

Thanks Nick.

My original Skyline file goes back to v22.2 and I see the same behavior on that version, so am unable to reproduce that aspect.

I've performed some more troubleshooting in Daily and noticed that I can fix the CE plot by removing and importing the file back in, but there is also a strange iRT display for the same plot which may be unrelated (showing iRT units instead of CE, e.g. iRT = 0.002 * Measured RT + 45.904). I've attached the before and after (re-importing the raw file), and this is all using the Collision Energy plot "Flat" that I made custom, as well as a screenshot comparison for the CE plot across the two files. Rescoring does not fix the CE plot, just reimporting fresh.

Cheers,
Chris

Chris,

Skyline remembers, for each Replicate, which collision energy equation was used at the time that time those results were imported.
In your "beforeReimport" Skyline document, the collision energy equation that is selected on the Prediction tab in Transition Settings is currently the one called "Flat". However, that only actually affects results that you may import in the future.
At the time that the results in that document were imported, the collision energy equation was something called "Thermo TSQ Quantiva" which specified a slope and intercept of both 1.
I believe the Skyline user interface does not give you any way to see what the equation is associated with each replicate. I am seeing this when I open the .sky file in a text editor.

Yes, it is confusing that when Skyline displays the equation on the Collision Energy Regression graph it says "iRT" and "Measured RT" instead of using terms related to collision energy. I suspect that it's always been like that and we just never noticed, but I'll try to figure out what is going on there.
-- Nick