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Spectral Library Build Error wootena  2024-08-22 07:15
 

Hello,

I am trying to build a spectral library using timsTOF data that was searched using Peaks Studio. I exported the db.pep.xml file from Peaks.

I am receiving an error message that reads ERROR index 10095 out of range in mzXML file ".\081023 C1 SDS 1 - 45 min_Slot2-1_1_1119.d.mzxml. I know this type of error was posted on the support forum previously and mentioned "this type of error was due to pep.xml file claims to have a spectrum match #, but there aren't that many spectra in the mzXML file."

I'm not quite sure how to fix this issue, but I would appreciate any feedback.

I have uploaded a folder with the error message, raw, mzxml, and pep.xml file to the file sharing folder for skyline support titled "Skyline Issue timsTOF" .

Thanks,
Ashley
 
 
Nick Shulman responded:  2024-08-22 07:24
Thank you for uploading that .zip file. I will take a look at that soon.

You cannot use .mzXML files with timsTOF data. The reason for this is that the .mzXML file does not have enough information in it to figure out which spectrum the results in the pepXML file are referring to.
Do you have any .mgf or .mzML files that you could use instead?

You might find some more information in the following support request which also refers back to other earlier support requests:
https://skyline.ms/announcements/home/support/thread.view?rowId=64727
-- Nick
 
wootena responded:  2024-08-23 07:24

Hi Nick,

Thank you for your response.

I moved the .mzXML files out of the folder and replaced them with the .mgf files.

I am now receiving a different message that reads " Could not translate integer 945 to native id format Bruker TDF nativeID format in .\081023 C1 SDS 1 - 45 min_Slot2-1_1_1119.d. Did not find spectrum 945 in .\081023 C1 SDS 1 - 45 min_Slot2-1_1_1119.d" I looked through the support requests and couldn't find a solution to the error message I received.

Thanks,
Ashley
 
Nick Shulman responded:  2024-08-23 07:42
It sounds like the .d folders are being used instead of the .mgf files.
I think you should delete those .d folders or move them somewhere else so that when BiblioSpec tries to find the mass spec data files that go with your .pep.xml it is only able to find the .mgf files.
It is also important that the .mgf files have the same name as the .d folders but with the ".d" replaced with ".mgf".
So, you should have a file called "081023 C1 SDS 1 - 45 min_Slot2-1_1_1119.mgf". If your .mgf files are not named like that then you might need to rename the files so that they have the correct expected names.

If that does not work then you should send me your .mgf files and I will figure out what is going on.
-- Nick
 
wootena responded:  2024-08-23 13:20

Hi Nick,

I made sure the files were named the same and I also moved the .d files, but it still didn't work for me.

I am uploading a zip file that includes the .mgf files into the file sharing named "Skyline Issues timsTOF MGF Files" The .mgf files are the original files, before changing the name to match.

I appreciate all of your help.

Thanks,
Ashley
 
Nick Shulman responded:  2024-08-24 18:34
Thank you for sending those .mgf files.
BiblioSpec does not want to use .mgf files to build the library.
I modified BiblioSpec so that it did use the .mgf files and I was able to successfully build a library, but I believe the library that I ended up (attached) with was nonsensical, where the spectra that were put into the library were not the ones that the peptide search engine intended to match.

Can you tell me more about how you did this peptide search? What file type did Peaks Studio actually search?
Can you maybe include some screenshots of the Peaks Studio settings, or command line parameters?

(I am not the expert on BiblioSpec, so it is possible that someone else will already know what the correct answer is or will have better questions to ask you)
-- Nick
 
wootena responded:  2024-08-26 06:03

Hi Nick,

These samples were searched using a human database. I have attached a screenshot of the parameters that were used for the search. The type of file that was searched was a .d file. I also attached a screenshot showing the file type for some of the files that were searched.

Thanks,
Ashley
 
wootena responded:  2024-08-30 05:46

Hi Nick,

I just wanted to follow up to see if there were any updates or if you needed more information from me.

Thanks,
Ashley