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!
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