Robert, If you haven't solved this already in the 2 weeks since you posted, you should focus on getting better scoring X!Tandem search results. For the two peptides of interest, OMSSA returns expectation scores of 1E-4 and 4E-6 (lower=better). But the X!Tandem expectation scores for these peptides range from 0.22 to values greater than 1.0. That's where your problem is, not in optimizing PeptideProphet run options. Figure out how to optimize your X!Tandem search parameters first.
On Aug 11, 7:34 am, Robert <[email protected]> wrote: > Hi, > > I´m using OMSSA and the TPP/X!Tandem search (+TPP PeptideProphet/ > ProteinProphet) for LC-MS/MS data analysis. > > First, I always run OMSSA due to its high performance to have first > information on the sample (for curious colleagues, and to set > priorities), then I make a more thorough analysis using the TPP. > > However, OMSSA reports some different peptides as the final TPP > output. > > When looking at the *.pep.xml, all peptides found by OMSSA, are also > present in the X!/Tandem-pep.xml file, but not reported at the end of > the pipeline. I already tried to set the p-filter for the > PeptideProphet to very low values (0.0001), but those additional > peptides do not show up. > > Example for the trypsin precursor: > > TPP reports: > ************** > > 7 gi|136406|sp|P06871.1|TRY1_CANFA gi|136429|sp|P00761.1|TRYP_PIG > 0.9565 > > confidence: 0.28 max coverage: 4.3% num unique peps: 1 tot > indep > spectra: 1 share of spectrum id's: 1.84% > > >RecName: Full=Cationic trypsin; Flags: Precursor Length: 246aa > > >RecName: Full=Trypsin; Flags: Precursor > > weight peptide sequence nsp adj prob init prob > ntt nsp total pep grp > ind > > * wt-1.00 2_LSSPATLNSR 0.9565 0.9851 2 0.00 > 1 > > OMSSA reports (top redundant hits): > ****************************************** > > Peptide: IITHPnFNGNTLDNDIMLIK Neutral Mass: 2283.3 Da > Charge State: > +3 E-value: 1.203E-006 > > Peptide: LGEHNIDVLEGNEQFINAAK Neutral Mass: 2211.5 Da > Charge State: > +3 E-value: 4.363E-009 > > In both searches I included deamidation of Q/N > > Searching those peptides in the TPP *.pep.xml gives: > ************************************************************* > > index spectrum hyperscore nextscore bscore yscore > expect ions > peptide protein > 1331 3D-1.01138.01138.3SpectraST 637 558 1 1 0.22 > 38/76 > R.LGEHNIDVLEGNEQFINAAK.IPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > 1339 3D-1.01143.01143.3SpectraST 622 595 1 1 0.71 > 34/76 > R.LGEHNIDVLEGNEQFINAAK.IPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > 1348 3D-1.01149.01149.3SpectraST 632 587 1 1 0.7 > 36/76 > R.LGEHNIDVLEGNEQFINAAK.IPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > 1400 3D-1.01180.01180.3SpectraST 697 649 2 2 0.23 > 42/76 > R.LGEHNIKVLEGNEQFINAAK.IPeptideAtlas gi|209572698|sp|P35030.2| > TRY3_HUMAN > > and > > index spectrum hyperscore nextscore bscore yscore > expect ions > peptide protein > 815 3D-1.00791.00791.2SpectraST 304 299 1 0 15 > 11/18 > K.IITHPNFNGN.TPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > 1472 3D-1.01223.01223.3SpectraST 569 497 1 1 0.65 > 33/76 > K.IITHPNFNGNTLDNDIM147.04LIK.LPeptideAtlas gi|136429|sp|P00761.1| > TRYP_PIG > 1490 3D-1.01234.01234.3SpectraST 559 530 1 1 4.9 > 30/76 > K.IITHPNFNGNTLDNDIM147.04LIK.LPeptideAtlas gi|136429|sp|P00761.1| > TRYP_PIG > 1545 3D-1.01268.01268.3SpectraST 585 555 1 1 4.2 > 28/76 > K.IITHPNFNGNTLDNDIMLIK.LPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > 1553 3D-1.01273.01273.3SpectraST 581 555 1 1 8.4 > 24/76 > K.IITHPNFNGNTLDNDIMLIK.LPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > 1565 3D-1.01280.01280.3SpectraST 531 522 1 2 7.5 > 32/76 > K.IITHPNFNGNTLDNDIMLIK.LPeptideAtlas gi|136429|sp|P00761.1|TRYP_PIG > > So, obviously, those peptides did not pass the PeptideProphet filter. > > ** Any recommendation, how to fix this to obtain a higher sequence > coverage in the TPP? ** > > Note: > If I tick the PeptideProphet Option " Force the fitting of the mixture > model (bypass automatic mixture model checks) " I increase the > sequence coverage of other proteins, but those trypsin fragments > still do not show up. > > Thanks a lot and greetings, > > Robert --~--~---------~--~----~------------~-------~--~----~ You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To post to this group, send email to [email protected] To unsubscribe from this group, send email to [email protected] For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en -~----------~----~----~----~------~----~------~--~---
