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/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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