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 <[email protected]> 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 <[email protected]> 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 <[email protected]> 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 <[email protected]> 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 <[email protected]> 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 <[email protected]> >>>>>>>>>> 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 [email protected]. >>>>>>>>>>> To post to this group, send email to >>>>>>>>>>> [email protected]. >>>>>>>>>>> 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 [email protected]. >>>>>>>>> To post to this group, send email to [email protected]. >>>>>>>>> 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 [email protected]. >>>>>>> To post to this group, send email to [email protected]. >>>>>>> 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 [email protected]. >>>>> To post to this group, send email to [email protected]. >>>>> 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 [email protected]. >>> To post to this group, send email to [email protected]. >>> 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 [email protected]. > To post to this group, send email to [email protected]. > 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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comet.params
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