Rugged extracted ion chromogram of targeted peptide precursors from FAIMS-DIA file by Skyline.

Rugged extracted ion chromogram of targeted peptide precursors from FAIMS-DIA file by Skyline. Winnie  2022-10-30

Hi, skyline,

I extracted chromotogram of targeted peptide precursors from FAIMS-DIA files by skyline, the xic show rugged profiling. The screenshot is attached. I was wondering there are issue on ion moblity library because i did not set ion mobility sepctral library. The sepctral library was imported by the following strategy. File>import>assay library. I found the column named precursor ion mobility in this spectral library is NA. The spectral library was build by Fragpipe with files acquired by FAIMS-DDA. I first communicated with Fragpipe team, they told me it is normal the column named precursor ion mobility is NA, because they this the FAIMS is low resolution device for ion moblity. So they set NA of precursor ion mobility for the library genetated by FAIMS-DDA.

So, did you have suggestion on the rugged xic and how to spectral library generated by FAIMS-DDA

Nick Shulman responded:  2022-10-30
When chromatograms look jagged like that in Skyline, it's usually because multiple different types of spectra are contributing to the extracted chromatogram points, and the different spectra have vastly different measured intensities.
In order to make your chromatograms smooth, you need to give Skyline some way of deciding which spectra should be included in the extracted chromatogram, and which excluded.

You can go to:
Settings > Transition Settings > Ion Mobility
and create a .imsdb file which will contain the compensation voltage values for each of your molecules. I think you can use the "Use Results" button in the Edit Ion Mobility dialog and Skyline will look at your jagged extracted chromatograms and figure out which compensation voltage worked best for each of your molecules and store those numbers in the .imsdb file so that the next time you extract chromatograms they will be smooth.

I am not sure where to find the best information about using Skyline specifically with FAIMS, but you will probably find the ion mobility tutorial useful:

-- Nick
Brian Pratt responded:  2022-10-30
Nick has you on the right track with Skyline's Settings>TransitionSettings>IonMobility>IonMobilityLibrary>Add>UseResults.

It's worth looking at this previous support thread, too:

And it's probably also worth thinking about how well FragPipe does with this data if it doesn't understand FAIMS ion mobility.

Best Regards,

Brian Pratt
Winnie responded:  2022-10-30
Hi Nick and Brian,

Thank you both for helping me. I have built the ion mobility sepctral library in the way you suggested (Settings>TransitionSettings>IonMobility>IonMobilityLibrary>Add>UseResults). I notice that if I used the UseResults option to build an ion mobility spectral library, the skyline would select the firstly imported raw data as the reference.

And I have another two questions about data extraction from FAIMS-DIA by skyline.

1. If I have one dataset which was aquired by slightly different compensation voltages such as -45v/-65v and -42v/-62v , It seems that I need to import those two kind of data by two skyline documents, right? Or I can directly use one skyline document to extract the data acquired by different compensation voltages.

2. Nick mentioned that skyline would use the own algorithm to filter the peptides that identified from multiple types of spectra. I would prefer to understand those multiple types of spectra maybe happen in different compensation voltages. For the peptides identified in different compensation voltages, how to select those by skyline algorithm? By intensity?

Best regards,

Brian Pratt responded:  2022-10-31
Hi Winnie,

1) Skyline does not yet support more than one mobility value per ion so yes, you'd have to go with two documents or turn off IM filtering and live with the jagged chromatograms.

2) Nick was probably referring to other instrument behaviors that can cause jaggedness (mode switch mid run etc) but in your case it's clearly just FAIMS at work. The algorithm is no mystery - you tell Skyline that a precursor ion is associated with a CV value, and Skyline will only use scans with that CV for chromatogram extraction on that precursor. If the question is "how does Use Result work?" then yes, Skyline just chooses the CV that yields the greatest intensity at the chromatographic peak that was derived before any ion mobility filtering was applied.

Best regards,

Brian Pratt
Winnie responded:  2022-11-01
Hi Brian,

I have built the ion mobility library by "UseResults". I notice that the Resolving power with different values shows a large difference. If I used the default value (Resolving power = 30), I could not extract any peak of the targeted peptide precursor. The raw file imported into skyline was acquired by standard mode (FAIMS PRO) in Exploris480 instruments. Skyline seems do not accept setting the resolving power with zero because if I set it to zero, I would be changed into a default number (Resolving power = 30)

For the data acquired by standard mode (FAIMS PRO), did you have any suggestion on resolving power of skyline for targeted peptide extraction?

Best regards,