Hello,

I am hoping to get some more information on the application of the q-value filter when setting up groupwise comparisons on the protein level in Skyline version 24.1. I am preparing to run pathway analysis on proteins that have been identified from DIA proteomics data in Skyline, so I am trying to generate high-quality groupwise comparisons with a p-value and log2 fold change for each protein. Implementing a q-value filter of 0.01 results in many missing abundances in my comparisons.

• From my understanding, the q-value essentially descibes the FDR for picked peaks. If it applies to individual peaks (ie, peptides), how is it calculated on the protein level?

• If I've already filtered my data by removing duplicate epptides, removing missing peaks, decoy scoring, etc. earlier in the analysis process, is it necessary to apply the q-value filter? Why is it applied in the groupwise comparison step and not earlier in the analysis process?

• Would you recommend applying the filter for this application?

Thank you for your help!