Command Failed_Return Code 65280

[xiany...@gmail.com]

One of my data failed and the return code 65280. The details are copied below.

I noticed that I have more decoys than non-decoys. However, I found many good MS2 spectra when I manually check my raw data.

What settings should I use to get the real good peptides validated through TPP (I used semi- when I did the database search using X!Tandem)?

Thanks.

Xianyin. 

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c:\Inetpub\tpp-bin\xinteract (TPP v4.6 OCCUPY rev 3, Build 201307241109 (MinGW))
 PPM mode in Accurate Mass Model ...

running: "C:/Inetpub/tpp-bin/InteractParser "interact.pep.xml" "20140307_12A.tandem.pep.xml" -L"7""
 file 1: 20140307_12A.tandem.pep.xml
SUCCESS: CORRECTED data file c:/Inetpub/wwwroot/ISB/data/20140307_12A.mzXML in msms_run_summary tag ...
 processed altogether 22005 results
INFO: Results written to file: c:/Inetpub/wwwroot/ISB/data/interact.pep.xml
command completed in 6 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/RAT20140219T_DECOY.fasta""
  - Searching the tree...
  - Linking duplicate entries...  - Printing results...

  - Building Commentz-Walter keyword tree...command completed in 9 sec

running: "C:/Inetpub/tpp-bin/PeptideProphetParser "interact.pep.xml" MINPROB=0.05 PPM ACCMASS DECOY=DECOY"
using Accurate Mass Bins
using PPM mass difference
Using Decoy Label "DECOY".
 (X! Tandem (k-score))
adding Accurate Mass mixture distr
init with X! Tandem (k-score) trypsin
MS Instrument info: Manufacturer: Thermo Scientific, Model: LTQ Orbitrap Velos, Ionization: electrospray ionization, Analyzer: radial ejection linear ion trap, Detector: electron multiplier

 PeptideProphet  (TPP v4.6 OCCUPY rev 3, Build 201307241109 (MinGW)) AKeller@ISB
 read in 0 1+, 6945 2+, 6638 3+, 4461 4+, 3930 5+, 0 6+, and 0 7+ spectra.
Initialising statistical models ...
Found 11360 Decoys, and 10614 Non-Decoys
Iterations: .........10.........20.........30..
WARNING: Mixture model quality test failed for charge (2+).
WARNING: Mixture model quality test failed for charge (3+).
WARNING: Mixture model quality test failed for charge (4+).
WARNING: Mixture model quality test failed for charge (5+).
model complete after 33 iterations
command completed in 34 sec

running: "C:/Inetpub/tpp-bin/ProphetModels.pl -i interact.pep.xml -d "DECOY""
Analyzing interact.pep.xml ...
Reading Accurate Mass Model model +1 ...
Reading Accurate Mass Model model +2 ...
Reading Accurate Mass Model model +3 ...
Reading Accurate Mass Model model +4 ...
Reading Accurate Mass Model model +5 ...
Reading Accurate Mass Model model +6 ...
Reading Accurate Mass Model model +7 ...
Parsing search results "c:/Inetpub/wwwroot/ISB/data/20140307_12A (X! Tandem (k-score))"...
  => Found 0 hits. (0 decoys, 0 excluded)
  => Total so far: 0 hits. (0 decoys, 0 excluded)
The system cannot find the path specified.
command completed in 1 sec

running: "c:/Inetpub/wwwroot/../tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/ISB/data/interact.pep.xml"

command "c:/Inetpub/wwwroot/../tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/ISB/data/interact.pep.xml" failed: Unknown error

command "c:/Inetpub/wwwroot/../tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/ISB/data/interact.pep.xml" exited with non-zero exit code: 255
QUIT - the job is incomplete

command "c:\Inetpub\tpp-bin\xinteract -Ninteract.pep.xml -p0.05 -l7 -PPM -OAp -dDECOY 20140307_12A.tandem.pep.xml" failed: Unknown error

[David Shteynberg]

All of your peptides have probabilities of 0 and since the setting shows MINPROB=0.05, all of the spetra are filtered from the resulting interact.pep.xml file.   This message is very telling "Found 11360 Decoys, and 10614 Non-Decoys",  assuming your decoy rate is 0.5 (rate of decoys amongst random matches) then you have 0 correct results in this search.  

-David

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[Brian Hampton]

Xianyin,

This recently happened to me.  In my case it turned out that the majority of proteins in the sample were not present in the search database.  Once I determined this and added in the correct species into the original search database, xinteract completed successfully.  Only a few proteins were from the targeted species were present in the sample and the vast majority of proteins in the sample were from the background species.  Without the protein sequences from the "background species" in the search database the analysis by the Prophets failed.  

Brian Hampton
Protein Analysis Lab
Center for Vascular and Inflammatory Diseases
University of Maryland School of Medicine
655 West Baltimore Street BRB 7-018
Baltimore  MD  21201
V: 410-706-8207

[David Shteynberg]

Brian,

The fact that the analysis gives you no peptides when there are no peptides to be had should be classified as a WIN!  Otherwise, you could be misled to publish erroneous conclusions based on false positive "identifications".

Cheers,

-David

[Brian Hampton]

HI David,

I should have said, .... Prophets failed as they should have since no targets were in the database.  You are correct that it is indeed a win.

Brian