Problem performing iterative CE optimization with explicit collision energies

Problem performing iterative CE optimization with explicit collision energies vincentroyrichard  2020-06-10


I'm currently trying to perform iterative CE optimization for an MRM method on a QTRAP 6500+ instrument using the latest version of skyline daily. What I would like to do would be 3 rounds of CE optimization with successively decreasing step sizes (starting with the CEs derived from the linear equation). What I'm noticing is that for the second pass CE optimization, I can only export a transition list for CE optimization with the optimized CEs (ie not derived from the linear equation) when entering these CE values in the explicit collision energy tab in the documents grid. The problem is that when I'm reimporting the data from the 2nd pass optimization it doesn't "center" around the correct CE.

For example, after optimization of a single MRM transition, the optimal CE was determined to be 18.6. I then reanalyzed the sample with a method to optimize the transitions with 3 steps +- 2 (ie 12.6 , 14.6, 16.6, 18.6, 20.6, 22.6, 24.6 -with 18.6 still remaining the most sensitive) - however when I reimport the data from this into skyline it changes the center CE to 24.6.

Seems to be a bug? Any assistance with this would be greatly appreciated!

Thanks very much!

Vincent Richard

Nick Shulman responded:  2020-06-11
I am not sure I understand what steps I would need to do to reproduce the problem that you are seeing.

If I would see the issue when importing results, can you attach your raw file? (I guess that would be the .wiff and the .wiff.scan)
If those files are less than 50MB, you can attach them to this support request.
Otherwise, you can upload them here:

Or, would I see this problem when I export a transition list? What settings are you using when you export the transition list?
-- Nick
vincentroyrichard responded:  2020-06-12
Hi Nick,

You can see this after data import. When you import the data optimizing CEs it basically changes the center CE at this point (from 18.7 -> 24.7 , you can see this in the results grid if you turn on "Opt Collision Energy"). When I tried to create an optimization library from results, it also takes the incorrect CE and thus affects the export of subsequent transition lists/methods when using optimization values.

I've attached the raw data (because we collect data as "nested" data files, please use the replicate labelled "xxxxx CE opt 2 m1").

Thanks very much for taking the time to look into this!
Nick Shulman responded:  2020-06-12
When I import results from this .wiff file, the "Opt Collision Energy" in the new replicate ends up being blank (i.e. "#N/A").
Can you attach the for what you get after importing this replicate? I'll try to figure out what I'm doing different.
-- Nick
vincentroyrichard responded:  2020-06-12
Hi Nick,

I've attached it post-import.

Nick Shulman responded:  2020-06-12
When Skyline reads chromatograms from a result file, Skyline does not know what collision energy was actually used.
Skyline just uses the equation from the collision energy predictor at:
Settings > Transition Settings > Prediction > Collision Energy

Skyline plugs the precursor m/z value into that equation, which is how it gets 24.7 as the collision energy for a precursor whose m/z is 438.7323++.

You have an optimization library "BAK270_CE_OPT_v1.optdb". You can see the values that are in there by going to:
Settings > Transition Settings > Prediction > Optimization Library > Edit Current

That library says that the collision energy for LYNPDVR++/y5+ is 18.65.
I do not believe that there is any way to see that number (the collision energy from the optimization library) in the Document Grid. However, I believe that if you were to export a method, that's the number that would be used.
(It looks like you might have typed in the number 18.65 as the "Explicit Collision Energy" for that transition in the Document Grid, and that's the number that would probably get used, but if you were to blank out the Explicit Collision Energy, then Skyline would use the value from the optimization library).

I am not an expert on CE optimization libraries, so I am not sure whether there is some step that should have been done differently.
Hope this helps,
-- Nick
vincentroyrichard responded:  2020-06-23
Hi Nick, sorry for the delay in responding! That is exactly the issue, when I export a new transition list specifically for CE optimization it doesn't take the value from the optimization library, but rather 24.7 even though I have "use optimization values when present" selected in this case.