FAIMS CV data analysis

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FAIMS CV data analysis Keri  2021-03-31
 

Hopefully a very straightforward question. I'd like to pick the best CV for each peptide identified and have been trying to follow this prior post https://skyline.ms/announcements/home/support/thread.view?entityId=32e29c0b-edcc-1037-8a70-e465a393af15&_docid=thread%3A32e29c0b-edcc-1037-8a70-e465a393af15. However, when I go to peptide settings>prediction there isn't a ion mobility prediction button apparent. Is there another place to find this feature or a way to add it? Thanks!

 
 
Nick Shulman responded:  2021-03-31
I believe the things that you are looking for have moved to:
Settings > Transition Settings > Ion Mobility
or maybe:
Settings > Transition Settings > Prediction

This change happened in Skyline version 20.2

-- Nick
 
Keri responded:  2021-04-01
Thanks Nick,

The closest I've found is transition settings>ion mobility>ion mobility library. However, when I try to create a library, it just generates an empty library. On Settings > Transition Settings > Prediction, there is an option for compensation voltage, which I have added. However that does not seem to change the output. Anywhere else that I should look? Also happy to upload a file if that would help. I'm using skyline daily, so it should be up to date

Thank you!

Keri
 
Brian Pratt responded:  2021-04-01
Hi Keri,

If you can provide your Skyline document (use File > Share to create a .sky,zip file) and the raw data file(s) involved, I'll take a look.

Best,

Brian Pratt
 
Keri responded:  2021-04-01
Thanks Brian! I've uploaded the zip file to the uploads page
 
Brian Pratt responded:  2021-04-01
Hi Keri,

Got the .sky.zip file, I'll need the raw data file as well.

Thanks

Brian
 
Keri responded:  2021-04-01
I've uploaded the mzXML (SY-50).
 
Brian Pratt responded:  2021-04-02
Looks like the problem is with the mzXML. I can see that the CoV values are in there but they aren't making it into Skyline for some reason. That's something we'll want to fix, but to get moving again just skip the mzXML conversion if you can, and work with the original mass spec output files. (That also makes for faster Skyline operation.)

- Brian
 
Keri responded:  2021-04-13
Hi Brian,

Just realized I never replied. The raw file worked. Thanks. Quick followup question. It seems like it would be really nice to be able to see/toggle through all of the CVs, as it appears that selection process for picking the best CV, while good, might not be 100% foolproof. Looks like others have asked this as well. Is there any way to visualize all of the CVs in a single experiment. My workaround has been to split the raw files and then process each CV separately, which is a bit unnecessarily complicated.

Thanks again,

Keri
 
Brian Pratt responded:  2021-04-14
Hi Keri,

I'm glad that worked out. It's pretty much always best to avoid file conversions when possible.

So you're wishing you could see the extracted chromatogram for each transition for each CV found in the data?

Best,

Brian
 
Keri responded:  2021-04-14
Yes it would be great to be able to see the chromatograms for each CV and/or quantify the data from multiple CVs for cases where peptides are shared between CVs.
 
Brian Pratt responded:  2021-04-14
Also, are you aware of Skyline's automated CV optimization capability?
 
Brendan MacLean responded:  2021-04-14
Which brings us full-circle to Nick's original response where he mentioned:

Transition Settings - Prediction

The "Compensation voltage" field in this form is definitely intended to be used with Thermo FAIMS and SCIEX SelexION mass spectrometers for SRM/MRM and PRM/MRM-HR. Optimized values for compensation voltage can also be stored in an "Optimization library" also accessible on the same settings tab.

Skyline is certainly capable of distinguishing between CVs, and if you collect your data in the right way Skyline can show you chromatograms for multiple CVs, exactly as it does for CE optimization described in this tutorial:

https://skyline.ms/tutorial_optimize_ce.url

Well, we are working on this for PRM, but it definitely works for SRM/MRM.

With Skyline, however, you might be able to add the peptides multiple times to your document, and set the Explicit Compensation Voltage to each value you are using on the multiple instances of the same peptide. And if that didn't work, you could have multiple files. But, you shouldn't need to break up the raw data file. Skyline is capable of extracting from just a specific CV, as long as you give it enough information.

--Brendan
 
Keri responded:  2021-04-14
Thanks-yes I did see that a while back, but have not been able to figure out how to implement it for our data. One weird thing I just noticed is that it seems that every time I close and open the skyline file I have to reimport the ion mobility library otherwise the data for all three CVs shows up. Any ideas for how to prevent this from happening?
 
Brian Pratt responded:  2021-04-15
Hi Keri,

I'm not sure I understand what you're describing - can you send:
 a Skyline document (use File>Share>Complete) saved while things are working properly
 a copy of that same Skyline document (use File>Share>Complete) that's in the state you're describing, before you do anything about reloading etc

Thanks
Brian