export a method with each transition CE optimized tongzhang  2020-03-12 13:47
 

In a project to optimize CEs for each transition, I created methods files with the option of optimizing collision energy selected. I ended up with 50 injections for 391 transitions on a TSQ. I followed a Skyline tutorial (link below) to upload the raw data, and would like to export a method with the best CEs for each transition. I enabled the "use optimization values when present" option, but can not get the optimized CE exported. One thing I noticed is that a schedule method was used in the tutorial, but I still want to export multiple methods because my peptides elute in a pretty narrow window.
I also attached the skyline files if that helps.
Thanks very much!

https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/OptimizeCE-1_4.pdf
 
 
Brendan MacLean responded:  2020-03-12 13:54

To get per-transition optimized values you need to choose "Transition" from the Optimize by dropdown list, which should appear below the "Use optimization values when present" checkbox when it is checked. By default "Precursors" is chosen, but if you make this change you should get what you are requesting.

You could also consider building an optimization library by choosing <Add...> form the Optimization library dropdown list above the checkbox. This would allow you to store the optimized CE values by transition for later use in documents that lack the full 50 injections you have created and imported into the current document.

Hope this helps. Thanks for posting to the Skyline support board.

--Brendan
 
tongzhang responded:  2020-03-12 14:18

Hi Brendan,
Thanks very much for your quick response!
Following the "Use optimization values when present" with "Transition" option, this is what I did for export. (please correct me if I did something wrong)

  1. Export --> Transition List;
  2. Instrument type: Thermo;
    Multiple Methods;
    Max transitions per sample injections: 100
    Optimizing: None
    Method Type: standard
  3. Click OK
  4. Choose YES to the following Skyline popup message:
    "The settings for this document do mot match the instrument type Thermo: Collision Energy:None Would you like to use the default method?"

In the method file I obtained, column 3 are values like -2, -4, 1, 2, and etc. I suppose that are values away from the initial prediction, right?
Before importing this method to TSQ, should I use the predicted value to calculate the actual CE values?
Thanks again!
T
 
Brendan MacLean responded:  2020-03-12 21:27

That sounds like a bug on our side, but it also just sounds like a bad idea to try to do this without using a document that is set to the CE prediction function you used in generating your original optimization methods. That is kind of an assumption of the code, at least until you get everything stored in an optimization library. It is not really valid to tell Skyline you want to use "None" as your CE prediction function, and Skyline should refuse to accept that outright.

You need to use the same CE prediction settings you used to generate your original methods for the 50 injections. All that gets stored in the resulting data files is the offsets from the original predicted values. If you do tell Skyline what you used to predict those values, then it will do the math for you and give you valid CE values in your exported method.

Again, if you want to forever store the actual CE values without needing to refer to the original predictor, you should build and optimization library. For now, you need to be careful about the steps you follow to make sure you don't go setting the CE predictor to something else or definitely not "None" between when you exported your optimization methods and when you try to export your optimized method.

Hope that helps to clarify. Thanks for explaining your situation.

--Brendan