FAIMS unit on Thermo Lumos

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FAIMS unit on Thermo Lumos erik.soderblom  2019-11-20
 

Hi Skyline Team! I wanted to "bump" a thread entitled “Optimizing FAIMS on new Thermo instruments” from Martijn van Duijn back in September. We are now in the same boat (acquired a shiny new FAIMS device for our Lumos!) and at this point we just want to incorporate the data (say, 3 different CVs across 7 different peptides) into Skyline from our normal system suitability PRM acquisitions. I see where compensation voltage is a selectable unit under the ion mobility settings, but it doesn't look like Skyline is able to extract data for a single ("best") CV in the chromatogram view. Is this possible with current Skyline Daily (19.1.1.309) yet?
Thx,
Erik

 
 
Brian Pratt responded:  2019-11-20

Hi Erik,

If I understand what you're asking for, you'd like Skyline to be able to automatically determine which available CV is ideal for each precursor, then ignore the other CV ranges when extracting chromatograms.

Skyline does have this capability (see the tutorial at https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ims ) although I am not certain that it has been used with FAIMS data yet. Have you tried it? If you find it doesn't work then let's get your data in here and we'll see what needs to be done.

Best,

Brian Pratt

 
erik.soderblom responded:  2020-04-20

Hi Brian,
Its been several months since this was brought up, but wanted to bring it back up once again. I was aware of the IMS capabilities within skyline, but i dont think they work well for FAIMS because a) it isnt a scanning device and b) there may be up to 3 different distinct compensation voltages (CVs) within an MS1 scan cycle (i.e. ms1 at CV1, ms1 at CV2, and ms1 at CV3). For our proteomics (and system suitability work) work, we typically define 2 or 3 distinct CVs so when you extract full MS1 data back into Skyline it "drops out" for the CVs which it does not exist. I've attached an image. It would be helpful to be able to select a single CV to show in the viewer (or allow quant over one CV) even if the instrument cycles between 3 of them in the instrument method. Note that this isnt a problem for any FAIMS-MS2 PRM data because you define a specific CV for each of those targets so it only extracts the data from one CV.
Cheers,
Erik

 
Brian Pratt responded:  2020-04-20

Hi Erik,

If you enable the "Explicit Compensation Voltage" column in the Document Grid, you will be able to set a CV value for each target. You can also specify this as part of a small molecule transition list.

Let me know how that works for you!

Best,

Brian

 
erik.soderblom responded:  2020-04-20

Hi Brian,

Thank you for the very quick response. That worked perfectly for our system suitability file and I can easily see how you would implement that for a targeted analysis! Thank you! I am, however, still curious about how we would implement this if we were working from a discovery experiment in which FAIMS-DDA data (say with 3 CVs) was acquired, searched, a library built within skyline, and the data imported to look at MS1 intensities. Does skyline read the header such it knows which CVs the (likely 100's-1000's) of peptides were identified from and then only plot that?
THx again for you help!

Erik

 
Brian Pratt responded:  2020-04-20

If you associate the CV value with the precursor as previously discussed, that should end up in the spectral library you build with Skyline for use in future chromatogram extractions.

If you don't actually know which CV value goes with which precursor, which I think is what you're talking about with a discovery search, Skyline can look through your data and determine which ion mobility value gives the largest chromatogram peak. Or, I think it can - that code was developed with continuous IM data in mind (e.g drift time and inverse reduced ion mobility) but it should work for CV data as well. If not we can have a look at it.

Best,

Brian

 
Brian Pratt responded:  2020-04-20

I should have mentioned that this is covered in the "Training a Drift Time Predictor" section of the Ion Mobility tutorial at https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_ims

Obviously we're not talking about drift time (or prediction, for that matter - that's a misnomer we're in the process of reworking) but the principles apply.

Cheers

Brian

 
Chris Ashwood responded:  2020-04-21

Hi Brian and Eric,

I can confirm that this works. I have used Skyline daily for data acquired on a Thermo Eclipse with a FAIMS Pro across a wide range of CVs (0-100 with 18 CV steps) for a single run. Initially it looked jagged due to incorporating each CV in the EIC of the precursor however when I went into the Edit Ion Mobility Predictor window, under molecule settings, and clicked Use Results. It autopopulated with the most intense CV for each small molecule. Attached is an example of the steps.

I have prepared the Skyline assay with audit logging enabled from start to finish so I'm happy to share if it helps someone.

Cheers,
Chris

 
erik.soderblom responded:  2020-04-21

Awesome! Thank you both!
Cheers,
Erik