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Ion mobility settings using timTOF Bruker DDA data

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Ion mobility settings using timTOF Bruker DDA data Luisa Nieto Ramirez  2024-01-23 10:48
 

Hello
I am currently developing a PRM method for a timsTOF Flex Bruker instrument. I have some questions that I hope you can help me with:

  1. In the SOP provided by Bruker, using Skyline to export the PRM method, they have under the peptide settings--> Prediction tab, the option for ion mobility predictor as shown in the first picture attached. However, in the current version of Skyline, we are not able to see this ion mobility predictor option. Do you know if that is no longer possible in the skyline versions after 2020? And what is used instead?

  2. We have been experiencing difficulties when trying to adjust the ion mobility isolation window to the actual values. We have seen the actual ion mobility values are shifted from the window set it by Skyline (see screenshot2). We tried to improve that, by manually integrating the peaks and then re-importing the data, however, it is still shifted (not working). That made us manually change the ion mobility window in the timsTOF software by looking at the heatmap observed in skyline. Do you have any suggestions on how to adjust the ion mobility window using skyline less prone to human errors?

  3. In the same line, under these ion mobility issues what would be the advantages or disadvantages of selecting "use spectral library ion mobility values when present" under the transition settings-->ion mobility.

This is a similar concern reported previously, but unfortunately, I was not able to generate a report with the "Explicit Ion Mobility Value" since the column for that variable was empty. https://skyline.ms/announcements/home/support/thread.view?entityId=f07ae1a2-f02b-1038-814c-e465a393b91e&_docid=thread%3Af07ae1a2-f02b-1038-814c-e465a393b91e

Thanks in advance for your help and time.

Luisa Maria
 
 
Brian Pratt responded:  2024-01-23 11:04

  1. The term "ion mobility predictor" wasn't really apt, it's now "ion mobility library" and can be found in the Ion Mobility tab of the Transition Settings window.

  2. The ion mobility library is the place to adjust Skyline's ion mobility values.

  3. It's just a way to ignore ion mobility values in spectral libraries in the event that you don't want to use them - perhaps you have an ion mobility library, or are setting per-precursor explicit ion mobilities.

Have a look at this tutorial for more information: https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ims

Thanks for using the Skyline support board,

Brian Pratt
 
Juan C. Rojas E. responded:  2024-01-23 14:55

Hi Luisa,

I would just like to add an experience I had with a timsTOF Pro. Although it shouldn't, the actual 1/K0 values recorded for the ions might change in between calibrations (the actual tolerance that is accepted is something I did not get to ask Bruker so far). The drift is not too large, but it can be limiting when you use high resolving powers for the ion mobility extraction windows as you are using; Resolving Power = 80.

I would recommend checking on the ion mobility spectrum to see what average window size would work best for your analytes; I tended to use 30-40 resolving power when I was working with datasets measured on different calibration events.

Hope it helps!
Sincerely,
Juan C.