Unfortunately, Skyline does not know how to export a method for the Stellar which includes collision energy values, so you cannot use the "File > Export > Method" menu item to create a method which has the stepped collision energy values which you would need to do collision energy optimization.

However, if you managed to create your own method where the collision energy values were what they needed to be according to the Collision Energy Equation, step size and step count specified in the "Collision Energy" dropdown on the "Prediction" tab at "Settings > Transition Settings" then you would be able to import the data into Skyline and choose "Collision Energy" in the "Optimizing" dropdown in the "File > Import > Results" dialog.

-- Nick

Great, that worked. Thank you!

Is it correct that I can only import CE-optimized files with equal CE spacing and uneven numbers in a way that matches the skyline export settings?

Yes, the collision energy on the MS2 spectra have to be within 0.05 of the value that you get by plugging the precursor m/z and charge into the Collision Energy Equation and adding the step number times the step size.
You do not need to have the full complement of collision energy step values. Skyline will look at the collision energy values on the spectra and figure out which step numbers they correspond to.
Often it is not possible to acquire the full complement of collision energy step values because the starting collision energy value is too close to zero, and, therefore, some of the steps would have to have a negative collision energy value.

You can see the individual collision energy step chromatograms by choosing "View > Transitions > Single".
If you click on a point along one of those chromatograms Skyline will show you the spectrum that contributed to that point. You can look at the property sheet (wrench icon on the toolbar above the spectrum) for that spectrum and make sure that it is the correct spectrum and has the correct collision energy.

Skyline is not very good at giving you error messages when things go wrong with collision energy optimization, so, if you are having trouble getting this to work you can send us your Skyline document and some of your .raw files and we can figure out what is going on.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
The Share Document dialog gives you the option of including the .raw files in the .zip file, or you could send them to us separately.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick

Thanks Nick,

I got it working for CE on the Stellar using the workaround. I am struggling with doing the same with FAIMS CV optimization: When importing the results, it will only show me the area under the curve for the central CV value, but none for the CV steps. I this in principle possible at all?
edit: I just saw that skyline puts all CV steps into a single trace (/ area under the curve bar). I found this under the wrench in the spectrum (ion mobility). Maybe this comes from the bad peak resolution and time requirements for switching CVs?

Thank you!

Unfortunately, optimization of full scan data only works with collision energy, not compensation voltage.

Optimization of compensation voltage only works with SRM data.
--Nick

I think what it's actually showing you is the sum of all the CV values - and you're seeing jagged chromatograms, yes?

The "Use Results" button in the Ion Mobility Library editor will look for the best CV value for each precursor ion and populate an ion mobility library with that information. Once you've done that, re-import the data using your new ion mobility library and you should see clean chromatograms with proper areas reported.

- Brian

Hi Skyfall,

An addition to the advice by Nick and Brian, I wanted to share a workaround that I use that might help you as well. You can create specific molecules with a specific spectrum filter, and duplicate those molecules by however many CVs you're using with FAIMS. Then modify the spectrum filter for each molecule to represent each step in your CV. The biggest downside with this approach is that you need to set the spectrum filter on a per-molecule basis (as far as I know) which is laborious.

I've attached a picture of how this looks when I do CE optimisation on a full scan instrument, but I don't see why this same approach won't work for Compensation Voltage (there is an option for that in the spectrum filter).

As you can hopefully see in the picture, the molecule rank option shows the best performing CE for the given molecule as well as there being overlapping chromatograms for each CE. For compensation voltage optimisation, this approach may not be optimal, but it's something to keep in mind.

More detail about the spectrum filter can be found at the link below:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=SpectrumFilters

Cheers,
Chris