Thank you Luis for helping me out here, just to follow up, I did try somebody else's condition file, since it seems my condition file didn't specify the 'isotopic contribution'. Still the libra columns shown [unavailable]. Thanks again sun
On Aug 22, 3:30 pm, sunsun <[email protected]> wrote: > yes, all peptides have no Libra data. no measured signal at all. > here is what shown in the commend log: > _____________________________________________________________________________________________________________________________ > run_in c:/Inetpub/wwwroot/ISB/data/itraq; xinteract - > Ninteract.pep.xml -p0.05 -l7 -Op -Lconditionfordevelopkit.xml-1 c:/ > Inetpub/wwwroot/ISB/data/itraq/itraq_r1_2.pep.xml c:/Inetpub/wwwroot/ > ISB/data/itraq/itraq_r1_6.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_7.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_8.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_9.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_2.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_6.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_7.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_8.pep.xml c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_9.pep.xml > > C:\Inetpub\tpp-bin\xinteract.exe (TPP v4.2 JETSTREAM rev 1, Build > 200904071723 (MinGW)) > > running: "C:/Inetpub/tpp-bin/InteractParser "interact.pep.xml" "c:/ > Inetpub/wwwroot/ISB/data/itraq/itraq_r1_2.pep.xml" "c:/Inetpub/wwwroot/ > ISB/data/itraq/itraq_r1_6.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_7.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_8.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_9.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_2.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_6.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_7.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_8.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_9.pep.xml" -L"7"" > file 1: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r1_2.pep.xml > file 2: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r1_6.pep.xml > file 3: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r1_7.pep.xml > file 4: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r1_8.pep.xml > file 5: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r1_9.pep.xml > file 6: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r2_2.pep.xml > file 7: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r2_6.pep.xml > file 8: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r2_7.pep.xml > file 9: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r2_8.pep.xml > file 10: c:/Inetpub/wwwroot/ISB/data/itraq/itraq_r2_9.pep.xml > processed altogether 4041 results > > results written to file c:/Inetpub/wwwroot/ISB/data/itraq/ > interact.pep.xml > > direct your browser tohttp://localhost/ISB/data/itraq/interact.pep.shtml > > ...skipping 81 lines of output... > > direct your browser tohttp://localhost/ISB/data/itraq/interact.prot.shtml > > command completed in 42 sec > > running: "C:/Inetpub/tpp-bin/ProtProphModels.pl -i interact.prot.xml" > Analyzing interact.prot.xml ... > command completed in 1 sec > > running: "C:/Inetpub/tpp-bin/LibraProteinRatioParser > "interact.prot.xml" -cconditionfordevelopkit.xml" > command completed in 3 sec > C:/Inetpub/tpp-bin/InteractParser "interact.pep.xml" "c:/Inetpub/ > wwwroot/ISB/data/itraq/itraq_r1_2.pep.xml" "c:/Inetpub/wwwroot/ISB/ > data/itraq/itraq_r1_6.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_7.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_8.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r1_9.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_2.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_6.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_7.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_8.pep.xml" "c:/Inetpub/wwwroot/ISB/data/itraq/ > itraq_r2_9.pep.xml" -L"7" 5 sec > C:/Inetpub/tpp-bin/PeptideProphetParser "interact.pep.xml" > MINPROB=0.05 3 sec > C:/Inetpub/tpp-bin/ProphetModels.pl -i interact.pep.xml 2 sec > C:/Inetpub/tpp-bin/LibraPeptideParser "interact.pep.xml" - > cconditionfordevelopkit.xml 1 sec > C:/Inetpub/tpp-bin/RefreshParser "interact.pep.xml" "c:/Inetpub/ > wwwroot/ISB/data/uniprot_sprot.fasta" 37 sec > C:/Inetpub/tpp-bin/PepXMLViewer.cgi -I c:/Inetpub/wwwroot/ISB/data/ > itraq/interact.pep.xml 2 sec > C:/Inetpub/tpp-bin/ProteinProphet "interact.pep.xml" > "interact.prot.xml" XML 42 sec > C:/Inetpub/tpp-bin/ProtProphModels.pl -i interact.prot.xml 1 sec > C:/Inetpub/tpp-bin/LibraProteinRatioParser "interact.prot.xml" - > cconditionfordevelopkit.xml 3 sec > job completed in 97 sec > > Command Successful > _____________________________________________________________________________________________________________________________ > > here is the condition file for Libra > ______________________________________________________________________________ > <?xml version="1.0" encoding="UTF-8" ?> > - <SUMmOnCondition> > - <fragmentMasses> > <reagent mz="114.1" /> > <reagent mz="117.1" /> > </fragmentMasses> > - <isotopicContributions> > <contributingMz value="1" /> > <contributingMz value="2" /> > </isotopicContributions> > <massTolerance value="0.2" /> > <centroiding type="2" iterations="1" /> > <normalization type="-1" /> > <targetMs level="2" /> > <output type="1" /> > <quantitationFile name="quantitation.tsv" /> > <minimumThreshhold value="20" /> > </SUMmOnCondition> > > ______________________________________________________________________________ > > On Aug 21, 4:56 pm, Luis Mendoza <[email protected]> wrote: > > > This is odd. Does it show this [unavailable] value for all peptides? Or > > are there some with a measured signal? (you can sort the output by that > > column to make it easier, or filter in the PepXMLViewer. > > > Can you please re-run xinteract the same way you ran it last time, and paste > > the entire output of the command in a reply? Can you also include the > > condition file, so I can make sure it got generated the right way? > > > The next step after this will be to look at the pep.xml file itself. If you > > can open it in a text editor (wordpad or some such...) , can you see some > > libra information near the top of the file? It would look something like > > this: > > <libra_summary mass_tolerance="0.200" centroiding_preference="2" > > normalization="3" output_type="1" channel_code="114115116117"> > > <fragment_masses channel="1" mz="114"/> > > <fragment_masses channel="2" mz="117"/> > > [...etc...] > > > Moreover, further down the file, can you find (use search) tags that look > > like this? (there shoud be one per peptide id'd) > > <analysis_result analysis="libra"> > > <libra_result> > > <intensity channel="1" target_mass="114.092" absolute="28.920" > > normalized="1.008"/> > > <intensity channel="2" target_mass="117.098" absolute="29.594" > > normalized="1.032"/> > > </libra_result> > > Let us know what you find, > > --Luis > > > On Fri, Aug 21, 2009 at 1:03 PM, zeyu sun <[email protected]> wrote: > > > Thank you Luis, > > > Well, actually by checking the original spectrum, I would say the labeling > > > is quite successful, almost all peptides have 114 and 117 peaks. I use the > > > raw data search the database by Mascot, the Mascot reports also have > > > 114/117 > > > ratio for all my peptides. > > > > I use the condition generator in the Libra panel with in the TPP. I just > > > check the 114 and 117 and put the % number there for -2, -1,+1,+2 isotopes > > > and leave other as default. > > > > There is no warnings when I ran the XInteraction. the log shown > > > successful. > > > > Zeyu Sun > > > > 2009/8/21 Luis Mendoza <[email protected]> > > > > Hello, > > >> The first thing I would check is if there are any iTraq signals in the > > >> spectra from a few peptide identifications. Click on any "ions" link in > > >> the > > >> pepXML Viewer, and then click on the "zoom 112-122" checkbox and "GO" > > >> buttons on the spectrum viewer that pops up. Can you see any of the > > >> reagent > > >> peaks? Do this for a few spectra. > > > >> The other thing to look for is the correctness of the condition file; how > > >> did you generate it? Lastly, were there any errors or warnings in the > > >> output of the tools when you ran them? Sometimes it is hard to miss if, > > >> say, Libra gave a warning in the midst of all the other output. > > > >> Let us know what you find, and thanks for trying our tools. > > >> --Luis > > > >> On Fri, Aug 21, 2009 at 10:43 AM, sunsun <[email protected]> wrote: > > > >>> This is my first time to use the TPP to process my iTRAQ-labeled > > >>> shotgun data, everything went successful until I found the > > >>> quantification of the iTRAQ data is [unavailable] in the result sheet > > >>> of 'Analyze Peptides', I have no idea why this happened and perhaps > > >>> someone here can give me some hint. > > >>> Here is the detailed data description and how I load them into the > > >>> TPP: > > >>> I only used 114 and 117 label from a iTRAQ development kit, just for > > >>> method development. Labeled peptides was further separated by SCX > > >>> before loaded into RP-HPLC coupled with an Agilent Q-TOF. Spectrum of > > >>> each SCX fraction was in .d files and was further converted to mzXML > > >>> by Trapper. I also converted .d files to mgf via Agilent's MassHunter > > >>> before the search. Database search was completed by Mascot and results > > >>> was exported in dat. format, which was further converted to pep.xml in > > >>> TPP. I put all pep.xml and corresponding mzXML files in one folder > > >>> under the directory of C:\Inetpub\wwwroot\ISB\data. In the TPP, I > > >>> selected the Mascot pipeline and loaded all the pep.xml files, > > >>> selected Run PeptideProphet and Run PeptideProphet afterwards. Run > > >>> libra using the condition file generated according to the isotopic > > >>> correction factors come with my kit. Everything else were leave as > > >>> default. > > >>> The results of running XInteract: both interact.pep.xml and > > >>> interact.prot.xml were generated. In the pepXML viewer, column 'pepP > > >>> probablity', 'spectrum', 'ionscore', 'identityscore','ions', > > >>> 'peptides', 'protein' all look good, but the Libra 114 and 117 columns > > >>> both shown [unavailable]. > > >>> Any comments, discussions are welcome!! > > >>> Thank you guys! > > --~--~---------~--~----~------------~-------~--~----~ You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To post to this group, send email to [email protected] To unsubscribe from this group, send email to [email protected] For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en -~----------~----~----~----~------~----~------~--~---
