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 <javascript:>> 
> 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*
>>>>>>>>>>>>
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