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*
>>>>>>>>>>>
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Attachment: comet.params
Description: Binary data

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