Thanks David! Let me try lowering fragment mass error. I will let you know about the outcome.
Well, we already had the Proteome Discoverer results and we really want to create the spectral library asap, so I was given those results to use instead of X!Tandem. and I have never worked with comet. I have to figure out how it works first. I will upload the ProteomeDiscoverer pep.xml in the folder that you have it's link, if you had a few minutes just take a look at it. May be it needed a few tweaks to work with PeptideProphet. Thanks so much, Ali On Wednesday, October 12, 2016 at 6:41:55 PM UTC-4, David Shteynberg wrote: > > The TPP is not tested to work with ProteomeDiscoverer pepXML. Sure you > can send me a sample file, but it may take some time to implement support > for this type of data. Comet is open source and easy to use out of the > box. Why don't you try that route? > > -David > > On Wed, Oct 12, 2016 at 3:24 PM, Ali <sma.ban...@gmail.com <javascript:>> > wrote: > >> Dear David >> >> Thank you very much! You were right, the problem was with X!Tandem. I >> decided do the searches with Proteome Discoverer. I exported the results to >> pep.xml (from .msf) using PD but now the results don't work with >> PeptideProphet. I can open them in PepXMLViewer but as I said in another >> post just now, I am having problem with processing them with PeptideProphet >> and InteractParser. Do you have experience with these files? I can send you >> a sample of the pepxml files for you. >> >> Sincerely, >> >> Ali >> >> On Wednesday, October 12, 2016 at 6:12:57 PM UTC-4, David Shteynberg >> 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.ban...@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/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-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-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-discu...@googlegroups.com <javascript:>. >> To post to this group, send email to spctools...@googlegroups.com >> <javascript:>. >> 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. 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