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Discrepancy issues with Peptide Search using .msf or .pdResult

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Discrepancy issues with Peptide Search using .msf or .pdResult Vera  2018-06-12 15:32
 

Hello Skyline team,

Currently, I analyze my data with Proteome Discoverer first and import the msf. file into skyline peptide search, then accept FDR<0.1 peptides only. However, recently my file is getting too big so I tried to import the pd.file (which suppose to contains only FDR<0.1 peptides only) instead. I noticed that less number of peptide and protein were identified when pd.file was imported (prot: 5689 pd. vs 8871 msf. ), but the number was not as small as I expected (about 50 protein). I was wondering is there any explanation for the difference between peptide search imported form msf. and pd. ? And is there any way to import FDR<0.1 peptides from Proteome Discoverer results (either msf. or pd.) only?

Thanks,
Vera
 
 
Brendan MacLean responded:  2018-06-13 09:26

Are you sure you are using FDR<0.1 (i.e. 10%) in both cases? The field seems to have settled on FDR<0.01 (i.e. 1%). I could imagine that the pdResult filter gives you this by default and you then use 0.9 (where 1 - 0.9 = 0.1 FDR cutoff) in the Skyline library builder, which would certainly give you more results from the MSF than the pdResult, but only because you used 10% FDR in MSF and 1% FDR in pdResult.

Percolator now also provides multiple levels of FDR:

  • PSM
  • Unique peptides
  • Unique protein groups

So, again, I expect that the FDR filter applied in producing the pdResult does not exactly match the cut-off applied during Skyline library building to the unfiltered MSF.

It will likely take you some digging to understand why. Hope my comments help.

--Brendan