Masses for charge state 1 are not appearing.

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Masses for charge state 1 are not appearing. PP  2025-05-22 12:31
 

Hello,

Could you please help me understand why I’m not seeing any masses for the +1 charge state, while I am seeing them for the +2 and +3 charge states?

 
 
Nick Shulman responded:  2025-05-22 12:46
The problem is that on the "Filter" tab at "Settings > Transition Settings", in the "Product ion selection" box, the choice in the "From:" dropdown is "m/z > precursor".
That means that Skyline is only going to give you transitions where the product ion m/z is greater than the precursor ion m/z.
This is Skyline's default setting, but, because product ions are always smaller than their precursor, it would mean that if your precursor charge is 1 you will not get any product ions.
If you are interested in charge 1 precursors, you might want to change "From:" to "ion 2" and "To:" to "last ion".

By the way, I see that on the "Full Scan" tab at "Settings > Transition Settings", the MS/MS Acquisition Method is "DDA".
Skyline is not a good tool for looking at DDA MS2 data. Skyline's primary purpose is to extract chromatograms, and chromatograms extracted from DDA data tend to have long straight lines connecting the times where a matching precursor happened to be selected for fragmentation by the mass spectrometer.
If you do want Skyline to extract DDA MS2 chromatograms for you, you will need to choose something in the "MS1 filtering isotope peaks included" dropdown on the "Full-Scan" tab at "Settings > Transition Settings". Skyline will only include a MS2 spectrum in the extracted ion chromatogram if the isolated precursor contributed to an extracted MS1 chromatogram, so you do need to tell Skyline to extract MS1 chromatograms if you want to see DDA MS2 chromatograms.

If you want to use Skyline with DDA data, I would recommend the DDA Search for MS1 Filtering tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_dda_search

-- Nick
 
PP responded:  2025-05-28 09:30
Thank you.