Hi Sayeef, I am forwarding your questions to the spctools-discuss forum since this is proper place to pose them and get the assistance you seek. Based on your account, I wonder if you have run PeptideProphet first? The workflow should be to create pepXML files from all your .out files (one pepXML file per msrun) (in answer to your question, dta files do not need to be deleted). Then all pepXML files should be combined and run through PeptideProphet to assign probabilities to all PSMs. Only then can you run spectrast to build a library. Does that perhaps help to solve the problem?
Regards, Eric > From: Mirza, Sayeef [mailto:[email protected]] > > Dr. Deutsch, > > We are using TPP for the goal of taking RAW data from the LC/MS-MS and > ultimately building a library to be used (imported into) Skyline. If > you could please help us with the few following questions, it would be > greatly appreciated. > > In following the general protocol for TPP. We first took the RAW files > and converted them to mzXML, successfully. Then we tried to doing > database search using TPP, but the amount of time involved was > impractical. Therefore we used the Sequest program and created directly > .OUT files from the RAW files. Actually the folder that was created > consisted of: .OUT files, .DTA files, and one .PARAMS file. Next, we > used that folder and converted it to a pepXML file. we used the same > parameters file. > > Lastly, we followed the directions for command prompt to produce the > .splib files. But the command prompt 'task summary' always shows "0 > peptides found, 0 signals above 0.9 prob" and also the resultant splib > file is only 1kb. That same splib file is not recognized in Skyline > where we are trying to build the library. > > Questions: > > * are we supposed to remove all the DTA files and only use the OUT > files + .params file for the converstion to pepXML? > * when running command prompt we have put both the mzXML file and > the pepXML file into the root directory of C drive, but still produces > a 1kb,unrecognizable splib file. > * When we checked the pepXML file using pepXML viewer, it shows a > legitimate list of peptides and they are all detailed and can be opened > as well, so it seems the pepXML file is fine. It is the step to make > the splib file that is not working. > > Essentially, this is what we've done to try and create a library from > our RAW files. Is there anything I should be doing different? > > Thanks, > > Sayeef Mirza > Moffit Cancer Center > Proteomics > Tampa, Fl > > > > > This transmission may be confidential or protected from disclosure and > is only for review and use by the intended recipient. Access by anyone > else is unauthorized. Any unauthorized reader is hereby notified that > any review, use, dissemination, disclosure or copying of this > information, or any act or omission taken in reliance on it, is > prohibited and may be unlawful. If you received this transmission in > error, please notify the sender immediately. Thank you. -- 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.
