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manual selection and change of ion mobility range for prm-PASEF

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manual selection and change of ion mobility range for prm-PASEF RBl  2020-10-14 02:45
 

Dear Skyline Team,
thank you very much for your constant efforts and all that you provide to the mass spec community! The software developed very much over past few years when i did not use it a lot!

I have started using Skyline in combination with the TimsTOF Pro recently. I needed to create a prm-PASEF method also with the ion mobility dimension. I noticed that ion mobility range that is showed in the Skyline was in many cases on the edge of the mobility peak/ion cloud and thereby a big proportion of the peak was not included. In some cases the ion mobility range in Skyline was in the middle of the mobility peak/ion cloud but edges were missing. I prepared some pictures so you can get the impression of what I am talking about. Please see the file attached.
I tried to set the ion mobility ranges in Skyline but I failed. Is there an option available for manual selection of ion mobility range or is it on me and I can not find it?
If there is no option for manual selection of ion mobility range, would it be possible to get something similar as manual chromatographic peak selection for Ion mobility peak/ion cloud in the future? And in the more distant future something like automatic peak peaking for ion mobility dimension if that is possible at all?

May you need more information from me, please do not hesitate to contact me.

Thank you very much for your efforts and your answer.
Best regards,
Renata
 
 
Brian Pratt responded:  2020-10-14 10:22

Hi Renata,

It sounds like you are using "Use Results" to identify the ion mobility peaks. Skyline's approach to this is pretty simple, it just chooses the IM frame that gives maximum signal at the chromatographic peak.

From your (excellent, thank you) report it appears that Skyline is doing a poor job of that. I would very much like to see your Skyline document (File > Share > Complete to create a .sky.zip file) and the raw TIMS data file, so that I can understand why this is happening.

Any file transfer method that is convenient to you is fine, just let me know. You can upload to http://skyline.ms/files.url , or email me at bspratt @ proteinms.net if you need to keep it private.

As to setting these values manually, you can specify an explicit ion mobility in a transition list, though the window size is still calculated per the settings in Settings > Transition Settings > Ion Mobility > Window Type. But ideally we'd just get those values right in the first place. (Note that this is difficult to do when there are a lot of interferences, though)

Thanks for using the Skyline support board,

Brian
 
Brendan MacLean responded:  2020-10-14 16:32

Hi Renata,
In the cases where the extraction range is fairly central, I would suggest that you just need a wider extraction range. It seems the resolving power you are using to determine the extraction range may be a bit too high. What resolving power are you using?

And, where are your ion mobility values coming from? Is these peptides or small molecules? (doubly charged at 700 m/z seems to imply peptides) Do they come from a spectral library built from ddaPASEF? If so, then they are where the DDA spectrum deconvolution software specified the precursor IMS value.

There is not a GUI interface to select the IMS extraction range, like there is to select the integration range in time for the chromatogram. Interesting idea, though. There is a report value "Explicit Ion Mobility Value" which you can set to override whatever your current source for ion mobility is. But, you will need to re-import your data, since this value only impacts chromatogram extraction during import.

But, it would be interesting to know the source of your ion mobility values which are ending up so far off of what appears to be the center of your peaks in the 1/K0 dimension.

--Brendan