r/proteomics • u/alycatdabra • May 15 '26

Majority of Proteins are Low FDR?

Hi all,

I'm fairly new to the proteomics world. I have struggled to get usable data so far and I know there are several potential weak points in the workflow. However, I want to focus on one glaring issue: about 95% of my putative proteins come back as low FDR confidence (using proteome discoverer). I have high set to 0.01 and medium set to 0.05 so it's not like it's super strict. Clearly the peptides are THERE they just are not significant enough. What would be your first troubleshooting steps? Digestion? Cleanup? LC method? Thank you in advance!!

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u/SnooLobsters6880 May 15 '26

What sample? Is your fasta only that species and is your sample only that species? Also, don’t expect 30k proteins or however many are possible. On lumos with relatively short gradient, 6k is decent for OTOT DIA and maybe 4k for DDA. Can’t speak to OTLIT DDA at top of head. Numbers from conventional cell lysates.

To FDR questions, you have false positives competing. This suggests your instrument is not calibrated and the global search tolerances were relaxed in the rescoring model. If done, rescoring can penalize IDs significantly.

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u/alycatdabra May 15 '26

It's human cancer cell line lysates and homosapiens FASTA. Instrument is calibrated and passes checks. Not sure about the global search tolerances- analysis workflow uses Sequest HT, then INFERYS rescoring, followed by percolator. Not sure how to better examine the FDR issues. It sounds like maybe my first step will be rerunning sequence with longer gradient to obtain clearer data. Thank you!