Thank you again Luis, There is quantitation.tsv file, but it shown 0 for all pepratio and -9 for protratio. I tried a new condition file with mass tel=0.5 and minimum threshold = 1 (in what unit??), and still its 0 for all pepratio and -9 for protratio. by the way, I check the mzXML files in the insilico view, most 114 peak's intensity are around 1500-2000....I believe somehow the TPP is not reading the intensity at all. zeyu
2009/8/24 Luis Mendoza <[email protected]> > Hello Sun, > The condition file seems correct for your set-up, as the 2 reagents you are > using are spaced by more than 2Da - so that the isotopic overlap is > negligible. Did you try altering some of the other parameters, such as a > wider mass tolerance, or a lower intensity threshold? It may be that your > reagent peaks have very small counts and are being ignored. Is there a > "quantitation.tsv" file created? If so, you can open it in Excel (or > equivalent program) and see what the raw counts are for the peptides. > > Cheers, > --Luis > > On Mon, Aug 24, 2009 at 2:03 PM, sunsun <[email protected]> wrote: > >> >> 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! >> > >> > >> >> >> -- Zeyu Sun Teaching Assistant, Ph.D Candidate @ Chemical and Biological Engineering, Colorado State University Glover 106, 1370 Campus Delivery, Fort Collins, CO, 80523 970-4911123 --~--~---------~--~----~------------~-------~--~----~ 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 -~----------~----~----~----~------~----~------~--~---
