PRM-PASEF method creation, 'All targets must have an ion mobility value'

PRM-PASEF method creation, 'All targets must have an ion mobility value' robert parker  2023-03-01 23:39

When creating a method for bruker timsTOF for PRM PASEF I get this message see attached.

The spectral library viewer shows the ionmobility values and I have it selected in transition settings, 'use spectral library ion mobility value when present'..

Is there some where else I need to specify this in skyline so it can creat the method?

bw and many thanks!


Brian Pratt responded:  2023-03-02 08:37

Could you perhaps share the Skyline document (use File>Share>Complete to create a file)? That's the quickest route to an answer.

Thanks for using the Skyline support board,

Brian Pratt

robert parker responded:  2023-04-06 09:16

Thanks Brian, heres the document attached.

I am sure its something simple I am doing wrong.

Brian Pratt responded:  2023-04-06 11:16

Have you tried doing the chromatogram extraction before exporting the method? I think there may be an unintentional dependency on that step creating the association between the target and spectral library IM value. We'll see about improving, but that would at least get you moving.

Please let me know if that helps.

Best regards,


Brian Pratt responded:  2023-04-06 11:28

Also, could you provide the template you were using? Once we get this sorted we can use that in an automated test to make sure this is fixed, and stays that way.



robert parker responded:  2023-04-06 13:25

Thanks Brian, appreciate the help. I did the extraction from the DDA data I have acquired for the library which is basically the heavy peptides. The same message pops up both when I try to export an isolation list or write method. You can see the ion mobility values in the chromatograms so I am not sure what's going on, I think I will just have to do this manually for now.

What do you mean by template, not sure what that is?

many thanks


Brendan MacLean responded:  2023-04-06 13:48

Hi Rob,
Can we get another file from you that includes imported data?

Looking at the code, it may be that we only support asking a spectral library for the ion mobility values for a specific file, i.e. for the DDA extraction case you just tried. At that point, you are asking the library for the IMS value for a file it knows about and it should use the correct IMS value.

I just don't see any code that knows how to get an IMS value from a spectral library without providing a file name. We will fix that.

In the meantime, you could turn around and train an IMS library from your imported DDA data. Building an IMS library from imported chromatogram data is explained in the Ion Mobility Spectrum Filtering tutorial:

Once you have an Ion Mobility library (.imsdb), then you definitely can use the values stored in it separately from the files those values came from.

Thanks for pointing this out. We will get the DDA -> PRM for prmPASEF working better soon.


robert parker responded:  2023-04-06 14:58

Many thanks for the help, I set up the calibration as described and now I can export the isolation list. Really appreciate the help.

robert parker responded:  2023-04-06 15:02

will share an example next week.

WeiQiang responded:  2023-06-20 01:29

I got the same error when I generated a spectra library using FragPipe ddaPASEF search result, imported a diaPASEF run of the same sample, and tried to export an isolation list for TimsTOF.

Do I need to import the same ddaPASEF raw data and use the spectroLibary before exporting the isolation list? The fasta for search is the whole proteome and I want to target only 12 proteins.

Juan C. Rojas E. responded:  2023-06-22 01:28

Sorry if I am too late to this discussion.

When the IM values are extracted from a spectral library does this automatically create an ion mobility library (.imsdb)? Or would this still have to be created through this path:

Settings -> Transition Settings -> Ion Mobility Library -> Add... -> Import... -> "A named spectral library"

Recently I have not been able to use this feature myself too much because the current database search and association software I use don't know how to report IMS. That said, I suspect that if you do it this way there will be cases where a precursor might have multiple IMS values (small decimal differences within the ion's IMS distribution). There will be cases where this fit to multiple gas phase conformers, but I think most instances are the former scenario and I saw that from fragpipe results about a year ago. I think we slightly touched on this on the Dortmund course.

@WeiQiang I have faced the error you had and the way I solve it is by tracking the precursor ions displayed like in the image Robert showed. But you can also create a custom precursor report (see attached figure) to track precursors that are missing IMS values. Perhaps you have some iRT peptides that are not reported in the spectral library?

If you only care for those 12 proteins, for the purpose of generating the method I would delete all other proteins and just keep those you care and make sure the associated precursors have reported ion mobility values.

Hope this helps.
And as always, thank you for your great work Skyline team.
Juan C.