Error Library Building from FragPipe (can't find mzml/mgf)

Error Library Building from FragPipe (can't find mzml/mgf) david schmidt  2024-04-16 04:41

Hi, in Skyline-daily (64-bit) (f9dd888b9), I tried to build a spectral library from FragPipe pep.xml output of a DDA-PASEF search. The error message indicates that Skyline is looking for a data file (mzml/mgf), which is actually present in the same folder. Any idea why Skyline doesn't recognize it? Noteworthy, the error message appears when clicking next on the spectral library tab (i.e. before selecting the raw data).

Thank you, David

Nick Shulman responded:  2024-04-16 10:46
I do not know what might be going wrong but if you send us your files we can try to figure it out.

You can zip up all of the files in that folder and upload them here:

I believe the name of mgf file that it is looking for is based on some filename that can be found inside of the pepXML file. It's not necessarily the same as the name of the pepXML file itself (although it often is the same name).

After we see your files I imagine we will be able to figure out what is going wrong.
-- Nick
david schmidt responded:  2024-04-16 23:48
Thank you Nick, I uploaded an example
Nick Shulman responded:  2024-04-17 08:04
For that file "3330m7Y_R3_ChymoDDA_231214_22min_373.pepXML", BiblioSpec would like to find a file that might be called "3330m7Y_R3_ChymoDDA_231214_22min_373_uncalibrated.mgf" or "3330m7Y_R3_ChymoDDA_231214_22min_373.mzML" (or any of a long list of other possibilities).

It would only be looking for something with "_uncalibrated" in its name if it ended in ".mgf". For the ".mzML" extension BiblioSpec is not trying to insert the "_uncalibrated".

You have a different pep xml file in there "interact-3330m7Y_R3_ChymoDDA_231214_22min_373.pep.xml" which thinks it was produced from a file called "3330m7Y_R3_ChymoDDA_231214_22min_373_uncalibrated" so it's going to be looking for files called "3330m7Y_R3_ChymoDDA_231214_22min_373_uncalibrated_uncalibrated.mgf" or "3330m7Y_R3_ChymoDDA_231214_22min_373_uncalibrated.mzML".

You might be able to get this to work if you use "interact-3330m7Y_R3_ChymoDDA_231214_22min_373.pep.xml" instead.

Alternatively, you might be able to get this to work if you rename some of the files that you have to what BiblioSpec is expecting. However, if you rename things, sometimes you are able to successfully create a spectral library but, because the wrong file type was used, and there was ion mobility involved, the spectra that were included in the library are the wrong ones. You can tell that this has happened because when you go to "View > Spectral Libraries" the spectra that you are shown have very very few fragment ions that match the peptide that was supposedly detected.

If you send me all of the mzML and mgf files you have I might be able to give you more information about what to do.
-- Nick