My suspicion regarding the Tandem failure is the fragment mass tolerance. I am observing fragment mass differences of much less than 0.05 daltons, yet your Tandem search uses 0.5 daltons as the tolerance. Could GPM-tandem be having a problem there? Can you try to lower the fragment mass tolerance and redo the search? Can you employ comet in your pipeline?
-David On Wed, Oct 12, 2016 at 3:12 PM, David Shteynberg < david.shteynb...@systemsbiology.org> wrote: > Hi Ali, > > I searched your data with Comet and PeptideProphet got valid PSMs in the > analysis that failed to produce correct results from your GPM-version of > tandem on the same data. The problem in your dataset is the Tandem > search. Attached are the comet parameters I used to search your data. > > > -David > > > > On Wed, Oct 5, 2016 at 9:36 PM, Ali <sma.banijam...@gmail.com> wrote: > >> I sent the link to your email. Thank you very much! >> >> On Wednesday, October 5, 2016 at 9:21:04 PM UTC-4, David Shteynberg wrote: >>> >>> I cannot tell from the search parameters if there is a problem. The >>> only issue I saw was that you didn't enable isotopic offsets, which are >>> sometimes helpful. If you send me a sample mzML file and possibly the >>> search results in pep.xml along with the database I can troubleshoot >>> further. One question, you said you are selecting 2+ 3+ and 4+, why are >>> only 2+ and 3+ in this dataset? Does any other dataset in your analysis >>> contain 4+? >>> >>> -David >>> >>> On Wed, Oct 5, 2016 at 6:16 PM, Ali <sma.ban...@gmail.com> wrote: >>> >>>> Hi David >>>> >>>> We are only collecting 2+, 3+ and 4+ data. I am working on the cluster, >>>> so I am using the GPM version and we were unable to install the k-score >>>> plugin therefore I am using the X! Tandem native scoring for the searches. >>>> >>>> If you mean combining all of the search results with xinteract and then >>>> using PeptideProphet (as you instructed), I have not yet done so because I >>>> wanted to make sure that we are getting results from the individual >>>> searches. Is there an issue in my search parameters which is responsible >>>> for the problem? >>>> >>>> Many Thanks, >>>> Ali >>>> >>>> On Wednesday, October 5, 2016 at 7:06:19 PM UTC-4, David Shteynberg >>>> wrote: >>>>> >>>>> Hi Ali, >>>>> >>>>> >>>>> Are you setting your instrument to collect only 2+ and 3+ data? Are >>>>> you using the tandem bundled with TPP or the GPM version of tandem? Have >>>>> you tried to process all your runs in one file? >>>>> >>>>> Thanks, >>>>> -David >>>>> >>>>> >>>>> On Wed, Oct 5, 2016 at 11:39 AM, Ali <sma.ban...@gmail.com> wrote: >>>>> >>>>>> Thank you very much! I really appreciate it! >>>>>> >>>>>> On Tuesday, October 4, 2016 at 8:03:27 PM UTC-4, David Shteynberg >>>>>> wrote: >>>>>>> >>>>>>> PeptideProphet is doing it's job here by telling you that there is >>>>>>> no distinct population of correct results in this search. I suspect >>>>>>> your >>>>>>> search parameters are the problem. This is orbitrap data so the charges >>>>>>> should be known, but it seems Tandem only searched 2+ and 3+ for you, >>>>>>> this >>>>>>> is somewhat troubling. Tomorrow, I will check your parameters more >>>>>>> carefully to try to identify the actual problem with your search. >>>>>>> >>>>>>> -David >>>>>>> >>>>>>> >>>>>>> On Tue, Oct 4, 2016 at 2:12 PM, Ali <sma.ban...@gmail.com> wrote: >>>>>>> >>>>>>>> It appears that PeptideProphet fails, I am getting this message: >>>>>>>> >>>>>>>> using Accurate Mass Bins >>>>>>>> using PPM mass difference >>>>>>>> (X! Tandem) (minprob 0) >>>>>>>> adding Accurate Mass mixture distr >>>>>>>> init with X! Tandem trypsin >>>>>>>> MS Instrument info: Manufacturer: Thermo Scientific, Model: >>>>>>>> UNKNOWN, Ionization: nanoelectrospray, Analyzer: orbitrap, Detector: >>>>>>>> inductive detector >>>>>>>> >>>>>>>> PeptideProphet (TPP v4.8.0 PHILAE, Build 201608051257-exported >>>>>>>> (RedHatEnterpriseServer-x86_64)) AKeller@ISB >>>>>>>> read in 0 1+, 14418 2+, 11080 3+, 0 4+, 0 5+, 0 6+, and 0 7+ >>>>>>>> spectra. >>>>>>>> Initialising statistical models ... >>>>>>>> negmean = 0.0533258 >>>>>>>> negmean = 1 >>>>>>>> negmean = 0.0376944 >>>>>>>> negmean = -0.2 >>>>>>>> negmean = -0.2 >>>>>>>> negmean = -0.2 >>>>>>>> negmean = -0.2 >>>>>>>> Iterations: .........10.........20. >>>>>>>> WARNING: Mixture model quality test failed for charge (2+). >>>>>>>> WARNING: Mixture model quality test failed for charge (3+). >>>>>>>> >>>>>>>> On Monday, October 3, 2016 at 5:57:39 PM UTC-4, David Shteynberg >>>>>>>> wrote: >>>>>>>>> >>>>>>>>> I highly recommend you start employing decoys in your search >>>>>>>>> databases. I noticed you are not combining together the runs in the >>>>>>>>> analysis. If the data was generated and searched in the same way it >>>>>>>>> should >>>>>>>>> be analysed together for better statistical power. I suggest you >>>>>>>>> combine >>>>>>>>> the results from all your runs and filter by the spectrum names >>>>>>>>> later. Try >>>>>>>>> this: >>>>>>>>> >>>>>>>>> InteractParser ./ALL.interact.xml ./*.tandem.pep.xml -S -Etrypsin >>>>>>>>> PeptideProphetParser ./ALL.interact.xml ACCMASS PPM NOICAT >>>>>>>>> MINPROB=0 >>>>>>>>> >>>>>>>>> >>>>>>>>> -David >>>>>>>>> >>>>>>>>> On Mon, Oct 3, 2016 at 2:39 PM, Ali <sma.ban...@gmail.com> wrote: >>>>>>>>> >>>>>>>>>> Hi David >>>>>>>>>> >>>>>>>>>> Thanks for your reply. I don't think so, it is not just one set, >>>>>>>>>> as I said almost a third of my search results are returned with zero >>>>>>>>>> probabilities. All of the searches are done with the same setting >>>>>>>>>> and using >>>>>>>>>> the same database. The fact that for some of the searched files I am >>>>>>>>>> getting zero probabilities and some of them are just fine is a bit >>>>>>>>>> strange. >>>>>>>>>> I don't have decoys in my data base. If it helps, I can send you one >>>>>>>>>> of the >>>>>>>>>> search results with or without PeptideProphet probabilities. >>>>>>>>>> >>>>>>>>>> Sincerely, >>>>>>>>>> Ali >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> >>>>>>>>>> On Monday, October 3, 2016 at 4:19:05 PM UTC-4, David Shteynberg >>>>>>>>>> wrote: >>>>>>>>>>> >>>>>>>>>>> Is it possible there are no correct results in that set? Common >>>>>>>>>>> pitfalls are incorrect search parameters (e.g. wrong mods enabled or >>>>>>>>>>> disabled) and wrong database. If you have decoys in your database >>>>>>>>>>> these >>>>>>>>>>> can be utilized for a more accurate mixture model generation using >>>>>>>>>>> options >>>>>>>>>>> "DECOY=<your decoy prefix> DECOYPROBS NONPARAM" in additions to the >>>>>>>>>>> options you already have enabled. >>>>>>>>>>> >>>>>>>>>>> -David >>>>>>>>>>> >>>>>>>>>>> On Mon, Oct 3, 2016 at 10:39 AM, Ali <sma.ban...@gmail.com> >>>>>>>>>>> wrote: >>>>>>>>>>> >>>>>>>>>>>> Hi >>>>>>>>>>>> >>>>>>>>>>>> I am using PeptideProphet on my X!Tandem search results. There >>>>>>>>>>>> are ~150 experiments and PeptideProphet works fine on most of them >>>>>>>>>>>> but for >>>>>>>>>>>> ~50 experiments, PeptideProphet returns zero probability for all >>>>>>>>>>>> of the >>>>>>>>>>>> peptides. Can some one please tell me what might be the issue? >>>>>>>>>>>> >>>>>>>>>>>> *More Info:* >>>>>>>>>>>> I am using TPP v4.8 on Linux. PeptideProphet does not show an >>>>>>>>>>>> error when it produces zero probability for all of the search >>>>>>>>>>>> results. >>>>>>>>>>>> This is what I'm doing: >>>>>>>>>>>> -search with X!Tandem >>>>>>>>>>>> -Tandem2XML ./100.tandem.xml ./100.tandem.pep.xml >>>>>>>>>>>> -InteractParser ./100.interact.xml ./100.tandem.pep.xml -S >>>>>>>>>>>> -Etrypsin >>>>>>>>>>>> -PeptideProphetParser ./100.interact.xml ACCMASS PPM NOICAT >>>>>>>>>>>> MINPROB=0 >>>>>>>>>>>> >>>>>>>>>>>> >>>>>>>>>>>> Thank you very much, >>>>>>>>>>>> *Ali* >>>>>>>>>>>> >>>>>>>>>>>> -- >>>>>>>>>>>> You received this message because you are subscribed to the >>>>>>>>>>>> Google Groups "spctools-discuss" group. >>>>>>>>>>>> To unsubscribe from this group and stop receiving emails from >>>>>>>>>>>> it, send an email to spctools-discu...@googlegroups.com. >>>>>>>>>>>> To post to this group, send email to >>>>>>>>>>>> spctools...@googlegroups.com. >>>>>>>>>>>> Visit this group at https://groups.google.com/grou >>>>>>>>>>>> p/spctools-discuss. >>>>>>>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>>>>>>> >>>>>>>>>>> >>>>>>>>>>> -- >>>>>>>>>> You received this message because you are subscribed to the >>>>>>>>>> Google Groups "spctools-discuss" group. >>>>>>>>>> To unsubscribe from this group and stop receiving emails from it, >>>>>>>>>> send an email to spctools-discu...@googlegroups.com. >>>>>>>>>> To post to this group, send email to spctools...@googlegroups.com >>>>>>>>>> . >>>>>>>>>> Visit this group at https://groups.google.com/grou >>>>>>>>>> p/spctools-discuss. >>>>>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>>>>> >>>>>>>>> >>>>>>>>> -- >>>>>>>> You received this message because you are subscribed to the Google >>>>>>>> Groups "spctools-discuss" group. >>>>>>>> To unsubscribe from this group and stop receiving emails from it, >>>>>>>> send an email to spctools-discu...@googlegroups.com. >>>>>>>> To post to this group, send email to spctools...@googlegroups.com. >>>>>>>> Visit this group at https://groups.google.com/grou >>>>>>>> p/spctools-discuss. >>>>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>>>> >>>>>>> >>>>>>> -- >>>>>> You received this message because you are subscribed to the Google >>>>>> Groups "spctools-discuss" group. >>>>>> To unsubscribe from this group and stop receiving emails from it, >>>>>> send an email to spctools-discu...@googlegroups.com. >>>>>> To post to this group, send email to spctools...@googlegroups.com. >>>>>> Visit this group at https://groups.google.com/group/spctools-discuss. >>>>>> For more options, visit https://groups.google.com/d/optout. >>>>>> >>>>> >>>>> -- >>>> You received this message because you are subscribed to the Google >>>> Groups "spctools-discuss" group. >>>> To unsubscribe from this group and stop receiving emails from it, send >>>> an email to spctools-discu...@googlegroups.com. >>>> To post to this group, send email to spctools...@googlegroups.com. >>>> Visit this group at https://groups.google.com/group/spctools-discuss. >>>> For more options, visit https://groups.google.com/d/optout. >>>> >>> >>> -- >> You received this message because you are subscribed to the Google Groups >> "spctools-discuss" group. >> To unsubscribe from this group and stop receiving emails from it, send an >> email to spctools-discuss+unsubscr...@googlegroups.com. >> To post to this group, send email to spctools-discuss@googlegroups.com. >> Visit this group at https://groups.google.com/group/spctools-discuss. >> For more options, visit https://groups.google.com/d/optout. >> > > -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To unsubscribe from this group and stop receiving emails from it, send an email to spctools-discuss+unsubscr...@googlegroups.com. To post to this group, send email to spctools-discuss@googlegroups.com. Visit this group at https://groups.google.com/group/spctools-discuss. For more options, visit https://groups.google.com/d/optout.
