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Missing Ion Mobility Values thomas poeschl  2025-01-03 04:28
 

Dear Skyline Team,

thanks for all the work you have put so far into your programm and community!
I am a young researcher and just starting in the field of Lipidomics.
Currently I have the challenge, that most of the Ion Mobility values which I generated from a standard lipid mix are not displayed in Skyline.
The measurements are done on a Bruker TIMS-TOF, this problem is found in both polarity modes.
For example, in my measurement the CCS value for [HexCer18:1/16:0+HCOO-] is displayed, while the Value for the same compound [M-H] is not. For further explanation a screenshot from Skyline is attached. So I looked this Ion up in the DataAnalysis software from Bruker and found the same ion mobility for the [M-H]-peak. Both Ion Mobilograms can be found in the second screenshot.
To work around this problem, I tried different resolving powers while importing under the tab Settings>Transition Settings>Ion Mobility.
So far, nothing worked, thats why I put my hope in you.
If needed, I will upload the skyline file as well.

Hope you have a nice day.

All the best,
Thomas
 
 
Brian Pratt responded:  2025-01-03 10:08

Hi Thomas,

Let's have a look at your Skyline document (use File > Share > Store Everything, and include at least one of your mass spec data files). Also please provide whatever artifacts you used to populate the Skyline document (transition list etc). You can upload to http://skyline.ms/files.url or we can arrange something private if you prefer.

Thanks for using the Skyline support board,

Brian Pratt
 
thomas poeschl responded:  2025-01-07 01:14

Hi Brian,

thanks for your fast reply!
I upladed the raw data for a lipid standard mix measurement, the first draft of a multitarget transition list (~200 lipids for following DDA experiments) and the Skyline file in the Zip folder "Ion mobility Skyline support_TP_07012025"
If you need more information, I'll be happy to do my best to find it.

Looking forward to your answer.

All the best
Thomas
 
Brian Pratt responded:  2025-01-07 09:32

Hi Thomas,

I see a few issues -

it looks like you didn't reimport the .d file after adding or creating the ion mobility library

the ion mobility library doesn't contain any value for some ions - such as those you mentioned. How was the library created?

And finally, it's DDA data, which is never great for chromatogram extraction of fragments.

If you have your own library CCS values, you could put those in the transition list and Skyline will use them as "explicit ion mobility values".

I hope this helps, don't hesitate to ask more questions. There's a lot I don't know about your process so it's hard to say where it's going wrong.

Best Regards
Brian
 
thomas poeschl responded:  2025-01-08 01:27

Hi Brian,

sorry, there seems to be a missunderstanding.
The library is an in silico generated library from LipidCreator, which only has MS1 and 2 spectra in it. The plan was to use MS1 and MS2 to annotate lipids, then update the RT and CCS values to build a 4D library for our purposes. I did not create any library by myself so far.
So I thought that I can measure the missing values (RT and CCS) to complete the library, or build it from scratch with all the data I will gain from the experiment. Isn't that possible?

Regarding the re-import: I re-imported to check, if that was the problem. Sadly, that did not help.

Maybe changing the measurement to DIA will help. I will try to measure DIA this week and will keep you posted, if the CCS values are visible in skyline. The original plan was to use DDA, since our biological samples would be to complex for unrestricted DIA. But with the right windows, I think DIA would be possible.

Thanks again for your input!

All the best
Thomas
 
Brian Pratt responded:  2025-01-08 11:32
 
Brian Pratt responded:  2025-01-08 11:33

Hi Thomas,

LipidCreator supplied that ion mobility library? I wasn't aware they had that feature. It's been some time since I looked at that tool - it really is excellent, though.

So it seems the issue here is just that Skyline hasn't been given the CCS values for some additional items. Skyline can help extend that ion mobility library to include the ions you've added, by looking for peaks in the IM dimension after chromatogram extraction without benefit of IM filtering - this tutorial should help illustrate the concept:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ims

Note that CCS determination is best done with low-complexity mixes, to avoid interference while seeking peaks in the IM dimension.

Cheers
Brian