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Skyline DIA quantification from DIA-NN

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Skyline DIA quantification from DIA-NN bseale  2026-03-23 06:37
 

Hello,

I was hoping to use Skyline to perform a 'traditional' quantification from DIA-NN data as opposed to its default MaxLFQ or highly questionable legacy mode.

I have managed to get everything loaded into Skyline, but, if I understand correctly, it seems that peptide summarization and later protein summarization is restricted to sum of some number (3? all of them? It's unclear) of precursors/peptides for DIA data. I was hoping Skyline would give me more control over this while still controlling some of the heuristics/logic in choosing which precursors/peptides to use across a data set.

Is my understanding correct? Am I forced to keep to sums and can't switch to something like median/mean?

Thanks for an excellent product,
Brendon Seale
 
 
Nick Shulman responded:  2026-03-23 07:51
If you want to compare quantities between groups of replicates, then Skyline does offer "Tukey's median polish" as one of the summarization options.
You can learn about group comparisons here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_grouped

However, even though Skyline does know how to do Tukey's median polish in a group comparison, Skyline does not know how to display that value in a report.
In terms of the "protein abundance" value that Skyline displays in reports, that will always be a sum of transition areas, and never a median.

You would need to export all the results yourself and then do your own median summarization outside of Skyline.
You can learn about custom reports here in the "Custom Reports" section on the tutorial page:
https://skyline.ms/home/software/Skyline/wiki-page.view?name=tutorials
-- Nick