Hi Laura,

When you look at the ion mobility heatmap, do you see a horizontal purple band indicating the IM range that was used in extracting the chromatogram? If not, you've not yet assigned a mobility value to your precursors, which is why nothing shows in the report.

If that's not the case, I'd be happy to look at your data (use Skyline's File > Share menu item, and include at least one of the mass spec files).

Thanks for using the Skyline support board,

Brian Pratt

Hi Brian,

I tried reimporting the runs and this worked, I see the purple band and the IM values are also showing in the report.
However, I noticed when doing this the peak areas are way lower than before, seems like something is going wrong with extracting the peaks when using an IM library. You can see an example in the attachment.

Laura

Also, I notice the IM and CCS it shows in every run is the same, this is probably the value from the library? How can I get the individual IMs for every run?

the peak areas are way lower than before

That's to be expected. Without IM filtering, Skyline uses all the signal across all IM values in the chromatogram extraction. With IM filtering, Skyline's chromatogram extraction ignores everything outside the target IM range (that purple band), yielding a more correct result with less interference. That is to say, that extra amplitude was coming from things other than the target.

the IM and CCS it shows in every run is the same

Again, yes, that's to be expected. Skyline is using the library CCS for each target to filter out signal from other potential co-eluting targets of similar mass and dissimilar CCS. I am working on a feature in which Skyline will say "you told me to look at IM range xxx and I did, but I noticed that the peak in the IM dimension is actually slightly off from the center of that range, by yyy".

Thank you for clarifying that, Brian!
Just one question, how does Skyline know which IM range to look at, or the one to build the library? I used the option "Use results" for the IM library.

Skyline just looks at the IM range you tell it to look at. This is the point of ion mobility separation in Targeted mass spec - you can tell Skyline about analytes of similar mass and RT that are indistinguishable unless you know to look in different IM bands, and you know which IM band pertains to which analyte. This can be done with an IM library, or values in a transition list, or in a spectral library.

When creating an IM library with Use Results, Skyline looks for the best peak in the IM dimension at the RT peak of the chromatogram that was extracted without benefit of IM filtering. Ideally you'd train the library on simple mixtures without interferences (co-eluting isomers etc) so that it can be used to deal with complex mixtures later, where IM separation really matters. Skyline is not completely naive in the presence of complex samples - it's understood that the IM value that yields the biggest signal at the RT peak might not be the IM value that actually belongs to the ion of interest, so Skyline considers isotopic pattern fit when deciding which IM peak to select. But this gets harder to discern as the sample complexity grows.

Note there are other sources of IM values out there, e.g. the Maclean lab's CCS Compendium or the Baker lab's PFAS CCS libraries. These CCS values have been obtained from simple mixtures so as to have high confidence in the measured CCS values actually belonging to the analyte of interest and not anything else that happens to co-elute and have similar mass.

I hope this makes sense, don't hesitate to ask further questions though.