Visualizing Multiple Collision Energy MS/MS Profiles in Skyline from a Single tMS2 File

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Visualizing Multiple Collision Energy MS/MS Profiles in Skyline from a Single tMS2 File dilip singh40288  2025-12-02 12:51
 

I am using Stellar MS to acquire tMS2 (PRM) data for a single compound at multiple HCD collision energies (e.g., 5, 15, 25, 35, 45). The output is a single RAW file containing MS/MS information at these different CEs. When I analyze the file in Thermo FreeStyle, I can view the MS/MS profiles for each CE and determine which collision energy works best.
However, when I try to analyze the same RAW file in Skyline, I cannot figure out how to visualize the MS/MS profiles at multiple CEs. A colleague suggested that to see fragmentation profiles in Skyline, I would need to acquire separate injections for each CE (i.e., five injections from the same vial, each at a different CE). I am wondering if there is a way in Skyline to view fragmentation profiles from a single tMS2 file that contains data acquired at multiple collision energies.

Looking forward for your response.

Regards,
Dilip Singh
CCHMC, Cincinnati

 
 
Nick Shulman responded:  2025-12-02 14:16
There are a couple of different ways that you could get this data to work with Skyline.

One way is to use the "Spectrum Filter" feature to tell Skyline that a particular Precursor in the Targets tree should only use spectra with a specific collision energy.
You can read about that feature here:
https://skyline.ms/home/software/Skyline/wiki-page.view?name=SpectrumFilters

A better way would be to set things up so that the collision energies that you acquired are the same energies that Skyline would have used when exporting a collision energy optimization PRM method. That would involve using the Document Grid to set the "Precursor Explicit Collision Energy" value to 25 and using the "Collision Energy" dropdown on the "Prediction" tab at "Settings > Transition Settings" to set the "Step size" to 10.

It might be tricky to get either of these ways to work, so the easiest thing might be for you to send us your data files and we'll get things set up for you.
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. Alternatively, you could send us the .raw files separately.

Files which are less than 50MB can be attached to these support requests. Larger files can always be uploaded here:
https://skyline.ms/files.url
-- Nick
 
dilip singh40288 responded:  2025-12-03 06:29
Thanks Nick,

Let me try suggested options! If works, good, if not, I will get back to you with raw file.

Regards,
Dilip