I think you need to set the "Explicit Collision Energy" values in the Document Grid for each of those transitions to the number that you want to use.

When you export a transition list and tell Skyline that you want to optimize collision energy, Skyline will never use the values from the .optdb file.
Skyline uses either the "Explicit Collision Energy" value that you may have specified, or it uses the Collision Energy Equation from "Settings > Transition Settings > Prediction > Collision energy".

The values from the .optdb file only get used if you are exporting a method that is not optimizing CE.

Manually adjusting the CE values after you export the method is not going to work right because Skyline does not know that you edited the CSV file. When Skyline later extracts chromatograms and sees that, for instance, Step -3 had the best signal, Skyline is going to assume that Step -3 had the collision energy that Skyline originally put into that CSV file.
-- Nick

Hi Nick,

thanks very much for your reply and your explanations. That's very helpful. I tried to play around in the Document Grid a bit, but couldn't quite figure out how to set the "Explicit Collision Energy" that you mention. Is there a Tutorial that shows how to do that, or would you send me some more details please?

Thanks again,
Regine

In order to set the "Explicit Collision Energy" in the Document Grid, you would need to first create a custom report that included the "Explicit Collision Energy" column.

I would do that by doing the following:
1. View > Document Grid
2. Choose "Transitions" in the "Reports" dropdown at the top of the Document Grid
3. Choose "Customize Report" in the Reports dropdown at the top of the Document Grid
4. In the Customize Report dialog, add the column "Explicit Collision Energy" to the report (there is a binoculars button on the toolbar at the top of the Customize Report dialog which lets you search for columns by name).
5. Set a Name for the report, and OK the Customize Report dialog.

There was no easy way for me to copy the collision energies "Edit Optimization Library" dialog into the Document Grid.
The document that you sent me only had six transitions belonging to a single precursor in it, so I could copy those transitions' collision energy values to the clipboard and then paste them into the Document Grid, but if you had a more complicated document that could have taken a while.

Note that I am not 100% sure that copying these values into the Explicit Retention Time column in the Document Grid is the recommended way of fine-tuning collision energy optimization because Skyline does not make it easy to copy the values from the .optdb file into the Explicit Retention Time column.

If you were trying to optimize compensation voltage for a FAIMS experiment, then Skyline has the concept of "rough", "medium" and "fine" sets of values in the .optdb file.

But, for collision energy optimization, it looks like we did not intend that the user would perform more than one round of optimization, or, if they did, it would only be in terms of multiple rounds of optimizing the linear equation at "Settings > Transition Settings > Collision Energy", not optimizing the values for individual transitions.

If you would like to learn more about the Document Grid, the "Custom Reports" tutorial has great information:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick