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Spectrum annotation inconsitencies

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Spectrum annotation inconsitencies jfoe  2024-02-14 05:10
 

Dear Skyline-Team,

I often try to identify peptides via skyline. In this effort, the "Show peak annotations" feature of the spectrum view can be really handy. But unfortunately, I really struggle to understand how it works, i.e. what leads it to match some peaks and not match others.

I am uploading two screenshots, one (spectrum_annotation_issue_control.png) shows how the transitions are matched from the canonical way of having transitions active in the targets panel. Note e.g. y7 and especially y8 are matched nicely between 900 and 1100 m/z.
Switching to the peak annotation view (spectrum_annotation_issue.png), I lose these matches (red box). I struggle to find any reason why this is so inconsistent.
The view is really helpful in many situations and e.g. in the example reveals the b3 match, which I didn't have enabled and I think it's a great feature!

Skyline version is 23.1.0.380 x64 on Windows.

Let me know if anything is unclear.

Best wishes,
Jonas
 
 
Nick Shulman responded:  2024-02-14 05:38
Can you send us your Skyline document and your raw file?
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 Skyline Document" also gives you the opportunity to include your .raw file(s) in the .zip file that you create, or you can send us the .raw file separately whichever is easier.

Files which are less than 50MB can be attached to this support request. You .zip file and/or the .raw file will probably be bigger than that so you should upload them here:
https://skyline.ms/files.url

When you first bring up the Full Scan graph by clicking on a chromatogram, Skyline shows you the m/z channels that were summed across to produce the extracted ion chromatogram. That is, for each Transition in the Targets tree, Skyline shades a portion of the graph showing you what part of the spectrum Skyline looked at to produce that transition's chromatogram. The width of the channel that Skyline highlights is controlled by the "Resolution" or "Mass Accuracy" setting at "Settings > Transition Settings > Full Scan".

When you choose "Show Peak Annotations", Skyline displays a label on top each peak in the spectrum that Skyline was able to identify as a particular potential fragment from your precursor, regardless of whether that transition actually appears in your Targets tree. In this peak annotation mode, the types of transitions that Skyline looks for is controlled by what has been chosen with the "Ion Types", "Special Ions", "Precursor" and "Charges" items on the right-click menu.
In order for Skyline to display the label on top of a peak in the spectrum, the observed m/z in the spectrum has to be sufficiently close to the calculated m/z of the fragment ion. The setting which controls how close those two numbers have to be is the "Ion match tolerance" setting at "Settings > Transition Settings > Library".

-- Nick
 
jfoe responded:  2024-02-14 05:57
Dear Nick,

I have indeed set an exceptionally narrow tolerance in "Ion match tolerance" setting at "Settings > Transition Settings > Library" due to how this usually applies to spectral libraries, which I generate in skyline, and which therefore have perfect mass accuracy.

I think knowing how this works now makes this completely tolerable for me and you have solved the issue for me.
I don't think this is documented anywhere else.
In principle, it would probably be great to have a mass tol. option right next to the ion selection in the right click menu, seeing how this directly applies and is quite crucial to the function.

I was hesitant to share the rawfile but let me know if it may still help you and I'll set it up.

Thank you for the great help and best regards,
Jonas