A low number of fragments are extracted, even if a high minimum product ion number is set.

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A low number of fragments are extracted, even if a high minimum product ion number is set. chiyy  2026-02-09 16:20
 

I am working on a PRM dataset. The Skyline file has been uploaded as 20260209_chiyy_LIT_GPF_survey_newAlign_MMCC_boundaries_opttrans_nochick.sky.zip. I found that the number of extracted fragments is relatively small (mostly 2–4), even after changing the settings to “16 product ions” and “10 minimum product ions” (see attached figure).

Are there any other settings I should adjust? Or does this indicate that only 2–4 fragments are available from the input files?

Thank you very much!

 
 
Nick Shulman responded:  2026-02-09 16:35
You should go to:
Refine > Advanced
and then check the checkbox next to "Auto select all ... Transitions".

For everything in the Targets tree, Skyline remembers whether the user made a change to which children a node in the tree has. If the user made a change, then it gets put into a state where the user is responsible for choosing which children that item has. The rules in the "Transition Settings" only apply to things that the user did not make changes to. If you right-click on one of the things in that tree and choose "Pick Children", there is a button which has a magic wand on it. When that button is selected, Skyline will apply the rules in Transition Settings to decide which transitions should be there. If the button is not selected, then you have to choose the children using the checkbox, or you could push the magic wand button to tell Skyline to use the Transition Settings rules.

If you check that "Auto select all ... Transitions" checkbox in the Refine > Advanced dialog then everything will get put back into the state where "Auto Select Transitions" is true.
-- Nick
 
chiyy responded:  2026-02-09 19:52
Thank you so much Nick. This really solved my problem. Now I can select as many transitions as I can.
 
chiyy responded:  2026-02-10 15:26
A follow-up question after I auto-selected the fragments.

I set the maximum number of product ions to 50 and the minimum product ions to 1. Theoretically, all fragments for each peptide should therefore be extracted, and indeed the list under each target tree is quite long. However, after exporting the chromatograms, I found that the number of fragments with XICs is still low, with a maximum of only 6.

Here is the distribution of fragment numbers:
{2: 810, 3: 25,272, 4: 132,624, 5: 30,915, 6: 40,662}.

I also checked the fragment areas in the Peak Areas view for each peptide, and many of them are 0. Does this mean that those fragments are truly flat at the baseline, or are there settings I can adjust to allow low-intensity (near-noise-level) fragments to have non-zero areas?

Since this is a PRM dataset, having a maximum of only 6 fragments per precursor seems a bit low to me. Thank you very much!
 
Nick Shulman responded:  2026-02-10 15:43
Did you tell Skyline to extract chromatograms again after you made the settings change that gave you more Transitions?

When you add Transitions to the document, you usually need to tell Skyline to extract chromatograms again which you can do by using the "Reimport" button at "Edit > Manage Results".

If the chromatogram had been extracted for these transitions but was completely flat, you would usually see the red symbol next to the transitions in the Targets tree. With there being no symbol at all next to them like in your screenshot, that probably means that the transitions were added to the document after chromatograms were extracted so the new transitions have no chromatogram at all.

There is an explanation of the meaning of the green, yellow and red symbols next to transitions here:
https://skyline.ms/home/software/Skyline/wiki-page.view?name=TargetsTreeSymbols
-- Nick
 
chiyy responded:  2026-02-10 15:58
Thank you very much Nick! I haven't done the reimport. I am working on the skyline file directly from Panorama. I am gonig to reimport from the raw files, and let you know if I have further questions.