Libra problem

[zeyu sun]

Dear TPP team,
I'm running a 8plex iTRAQ data analysis using the Mascot-TPP-Libra pipeline. It went through peptideProphet and proteinProphet smoothly without Libra. However, when I add Libra option into the picture, it generate an error. It seems the Libra quantitation require the spectrum data in mzXML format, however, all I have is mg files. Do I really need to convert everything to mzXML? or is there something else going wrong that I really unaware off. Please help me out!
thank you so much!
SunSun


here is the error message I got:

EXECUTING: run_in c:/Inetpub/wwwroot/ISB/data/2ndhospital/1; c:\Inetpub\tpp-bin\xinteract -Ninteract.pep.xml -p0.95 -l7 -O -Lcondition_zeyuiTRAQtest1.xml-1 F003158.pep.xml F003159.pep.xml F003160.pep.xml

c:\Inetpub\tpp-bin\xinteract (TPP v4.6 OCCUPY rev 0, Build 201208170820 (MinGW))

running: "C:/Inetpub/tpp-bin/InteractParser "interact.pep.xml" "F003158.pep.xml" "F003159.pep.xml" "F003160.pep.xml" -L"7""
 file 1: F003158.pep.xml
 file 2: F003159.pep.xml
 file 3: F003160.pep.xml
 processed altogether 2737 results


 results written to file c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/interact.pep.xml

 direct your browser to http://localhost/ISB/data/2ndhospital/1/interact.pep.shtml



command completed in 5 sec 

running: "C:/Inetpub/tpp-bin/DatabaseParser "interact.pep.xml""
command completed in 1 sec 

running: "C:/Inetpub/tpp-bin/RefreshParser "interact.pep.xml" "c:/Inetpub/wwwroot/ISB/data/dbase/zeyu_human.1.fasta""
  - Searching the tree...
  - Linking duplicate entries...  - Printing results...

  - Building Commentz-Walter keyword tree...command completed in 18 sec 

running: "C:/Inetpub/tpp-bin/PeptideProphetParser "interact.pep.xml" MINPROB=0.95"
 (MASCOT)
results for charge 1: 9 id tot and 1 adj scores
results for charge 2: 512 id tot and 16 adj scores
results for charge 3: 903 id tot and 46 adj scores
results for charge 3: 7.65044 adj_ion_mean and 23.2164 adj_ion_hom mean 37.4548id mean0.928281 correlation (r) 
3+ ion - id = 1.02138*(ion - hom) + -16.0625 with error = 6.00641
mean ion - id: 7.65044, mean ion - hom: 23.2164
results for charge 4: 347 id tot and 1 adj scores
results for charge 5: 966 id tot and 1 adj scores
results for charge 6: 0 id tot and 0 adj scores
results for charge 7: 0 id tot and 0 adj scores
init with MASCOT trypsin 
MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

 PeptideProphet  (TPP v4.6 OCCUPY rev 0, Build 201208170820 (MinGW)) AKeller@ISB
 read in 5 1+, 382 2+, 813 3+, 324 4+, 886 5+, 0 6+, and 0 7+ spectra.
Initialising statistical models ...
Iterations: .........10.........20..
WARNING: Mixture model quality test failed for charge (1+).
WARNING: Mixture model quality test failed for charge (4+).
WARNING: Mixture model quality test failed for charge (5+).
model complete after 23 iterations
command completed in 5 sec 

running: "C:/Inetpub/tpp-bin/ProphetModels.pl -i interact.pep.xml"
Analyzing interact.pep.xml ...
Parsing search results "c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003158 (MASCOT)"...
  => Total of 12 hits.
  => Total of 0 decoy hits.
  => Total of 0 excluded hits.
Parsing search results "c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003159 (MASCOT)"...
  => Total of 14 hits.
  => Total of 0 decoy hits.
  => Total of 0 excluded hits.
Parsing search results "c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003160 (MASCOT)"...
  => Total of 45 hits.
  => Total of 0 decoy hits.
  => Total of 0 excluded hits.
command completed in 5 sec 

running: "C:/Inetpub/tpp-bin/LibraPeptideParser "interact.pep.xml" -ccondition_zeyuiTRAQtest1.xml"
Failed to open input file 'c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003158.mzXML'.
could not open input file c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003158.mzXML

command "C:/Inetpub/tpp-bin/LibraPeptideParser "interact.pep.xml" -ccondition_zeyuiTRAQtest1.xml" failed: Operation not permitted

command "C:/Inetpub/tpp-bin/LibraPeptideParser "interact.pep.xml" -ccondition_zeyuiTRAQtest1.xml" exited with non-zero exit code: 1
QUIT - the job is incomplete

command "c:\Inetpub\tpp-bin\xinteract -Ninteract.pep.xml -p0.95 -l7 -O -Lcondition_zeyuiTRAQtest1.xml-1 F003158.pep.xml F003159.pep.xml F003160.pep.xml" failed: Operation not permitted

Command FAILED
RETURN CODE:256


The Libra condition.xml was generated within TPP using the default 8plex setting.

[ChargedPeptide]

Hey SunSun, I've had a number of problems running through mascot data. The most usual error for me is that the 

<msms_run_summary base_name=

in the pep.xml is set erroneously. This is especially true if you are running Mascot2XML on a merged Mascot search where results from multiple MGF files are in one .dat output file.

Libra reads the msms_run summary_base_name= and raw_data= to get the location and extension of input data. 

I've recently wrote a bit of Java to split the results from Mascot output files into their own pep.xmls after Mascot2XML and I'm not really aware of another way to do it, though I've missed something. 

If this is your problem I'd need to adapt it a bit (right now it only runs as part of our pipeline, not stand-alone) but I'd be happy to send it over if you need it.

Your first check should be if this: c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003158.mzXML is an actual data file. It looks like a mascot-search ID to me.

[zeyu sun]

Erik,
Thanks for the reply. However, the problem you described is not what I have encountered. I did the search separately without merging them, so I have individual dat. files that corresponding to each mzXML.In my case, the Libra have no problem to read the data at all. I can do the Mascot-TPP-Libra successfully, and I can also do the Tandem-TPP-Libra successfully, but I just haven't find a way to combine both data into a prot.xml. 

Despite of that, I think your Java script is very interesting and may help others if they want to try it out.
Thanks for the help.

SunSun

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Sun

[ChargedPeptide]

Oh, that's much easier :)
In this case you would need to do the following:
Run your mascot->pep.xml with libra quantities and also X!tandem -> pep.xml (If this is the same data ran with different search engines, if it is different data as well you need to process each batch separatley and set an experiment ID for each set, let me know if this applies and I'll give you som detail)

Run iprophet on both your output pep.xmls to get a pep.xml with combined spectral matches and iProbabilities.
Run protein-prophet on the iProphet output pep.xml.
Now you will have a prot.xml with no quantities displayed.
Manually run libraproteinratioparser (or similiar name, can't check now , on train)  as this:
libraproteinratioparser <your prot.xml> -c<your libra condition file>

Voila. Enjoy your final prot.xml.

Let me know if this works out for you.

[zeyu sun]

Erik,
I tried this before, but the libraproteinratioparser keep crashing all the time:
here is the error message I got when I try to use it to process the prot.xml:

This application has requested the Runtime to terminate it in an unusual way. Please contact the application's support team for more information.

any quick remedy to this problem?
thanks!

SunSun

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Sun

[Luis Mendoza]

Hello SunSun,
Libra does indeed need access to the spectra in mzML (or mzXML) format
to carry out the quantitation, as you pointed out from the error
message:

"Failed to open input file
'c:/Inetpub/wwwroot/ISB/data/2ndhospital/1/F003158.mzXML'"

It would be best if you could track down the original raw files for
your 3 runs and convert them to mzXML.

Cheers,
--Luis

[zeyu sun]

Thank you Luis, 

Yes, now I use the mzXML and the problem seems solved. 

SunSun

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Sun