Annotation of Light peptide fragments instead of heavy fragments

Annotation of Light peptide fragments instead of heavy fragments nyalwijo  2020-11-22

Dear colleagues,
I have PRM data for a SIL peptide. THe MS2 fragmentation data is perfect but Skyline annotates fragments for the Light peptide which are not in the MS2 spectrum instead of the Heavy peptide fragments. How can I correct the issue? Thank you. J

Nick Shulman responded:  2020-11-22
It looks like Skyline was not able to find any MS2 spectra that matched the 487.2213 precursor m/z of your heavy peptide.
You should look at your mass spec data file and make sure that there really are some MS2 spectra with that isolation window.
(I usually use a program called SeeMS.exe to look at mass spec data files. You can install SeeMS.exe along with the rest of ProteoWizard from You also might have your own instrument vendor software for looking at mass spec data files.)

Another thing that often happens when you are doing a Targeted acquisition in Skyline is that there are some spectra in the file which you intended to be associated with that precursor, but which Skyline is assigning to a different precursor. If the MS/MS filtering Acquisition method (specified at "Settings > Transition Settings > Full Scan") is "Targeted", then Skyline will assign any particular MS2 spectrum to whichever precursor had a m/z which was closest to what was isolated for that scan. If some of your MS2 isolation windows are intended to be used for more than one precursor, then you should change the Acquisition Method to "DIA", and set the Isolation Scheme to probably "Results Only".

If you are still stumped about what is going wrong, you can send us your Skyline document and one of your raw files.

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. If that .zip file and your raw file are less than 50MB, you can attach them to this support request. Otherwise, you can upload them here:

-- Nick
nyalwijo responded:  2020-11-23
HI Nick,
Thank you very much for your prompt response. My mass spec data file has the MS2 spectra with the isolation window and the target peaks are present and with good intensities. I proceeded as per your recommendations on your second point. i am running a targeted acquisition and I have changed the transition settings Acquisition Method to "DIA", and set the Isolation Scheme to probably "Results Only". This has resolved the problem and the heavy fragments are detected and assigned by Skyline. I would be very grateful if you could take a look at my skyline document and the raw file to uncover any other potential mitigating issues. Please let me know how I can send you the files privately. Best regards and I hope to hear from you soon.
Nick Shulman responded:  2020-11-23
Thank you for uploading those files.

Your "Method match tolerance m/z" at "Settings > Transition Settings > Instrument" is currently set to "0.0005" (the default value was "0.055").
This is the reason that Skyline was not matching your heavy precursors to their MS2 scans. The scans in your .raw file that were intended to be associated with 487.2213 were recorded by the mass spectrometer as having isolated 487.23029.

If you change the method match tolerance m/z to a higher number, then you can go back to saying that your acquisition method is "Targeted".

(I will send you an email directly in case there are other files you wanted to send privately)
-- Nick