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 -~----------~----~----~----~------~----~------~--~---
