Unable to see any fragment ions in Skyline

support
Unable to see any fragment ions in Skyline clgunth2  2020-10-13
 

Hello - I created a test transition list with ions I can see in the raw mass spectra and imported into Skyline. When I imported the data, only the precursor mass is identified with the green circle next to the precursor name, even though the ions in my transition list were extracted from the raw file. The fragment ions don't have a red circle for being not detected but rather say "Chromatogram not available" if clicked on. I'm not sure if it's just not reading my data correctly or not. Again, these fragment ions were chosen directly from the raw spectra to test the workflow in Skyline, so they are present in the raw data.

My data file contains only MS2 scans for one precursor. My goal is to be able to filter through a much longer list of theoretical fragments for one complex structure. I'm following similar to what I would do if I was filtering through a transition list with MS1 data except the data file contains solely MS2 scans.

Thanks,
Crystal

 
 
Nick Shulman responded:  2020-10-13
In your Skyline document, the only transitions that you have told Skyline to monitor are your precursor transitions. That is, the m/z of the transition is the same as the m/z of the precursor.

Skyline can extract precursor chromatograms from either MS1 or MS2 scans. Usually people want to see the MS1 precursor chromatogram since it has much better signal than monitoring the amount of intact precursor in the MS2 scans.

In order to tell Skyline to extract chromatograms from MS1 scans, you have to choose something at:
Settings > Transition Settings > Full Scan > MS1 Filtering > Isotope Peaks Included

Currently you have that set to "None", but if you change that to "Count" or "Percent", Skyline will know that you want to extract MS1 chromatograms again.

Whenever you change your settings or add Targets, you need to tell Skyline to extract chromatograms again. One way to do this is to go to:
Edit > Manage Results > Reimport

You probably also want to tell Skyline to monitor some MS2 transitions. You can add more transitions by right-clicking on the precursors in the Targets tree and choosing "Add Transition".

You should probably take a look at some of the tutorials on the Skyline web site. I would recommend the "Small Molecule Targets" tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials

I am not sure I understand what you are saying about the raw data file that you have. If you would like, you could send us that raw data file, and I could tell you whether your other settings are appropriate for that data. If that data file is less than 50 MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

-- Nick
 
clgunth2 responded:  2020-10-13
Hey Nick,

I think I had put all the m/z's of the product ions as precursor ions and that it why I couldn't see them! I just readjusted my transition list and can now see product ions in skyline!

Thank you,
Crystal Pace