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Unable to build PEPTIDE spectral library using .pep.xml and .mzxml files downloded together from PEAKS 11

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Unable to build PEPTIDE spectral library using .pep.xml and .mzxml files downloded together from PEAKS 11 ciaranmc 28  2024-06-05 11:51
 
Dear Skyline supporters,

I am attempting to build a spectral library using pep.xml and .mzXML files downsloded from PEAKS 11 (derived from DDA run on TimTOF, filtered for MS2 peptides at 1% FDR)

However, despite rerunning my peaks analysis to try again, I am repeatedly met with the skyline error included below (which suggest discrepancy between the pep.xml and mzXML files).

Both files were exported from PEAKS at the same time and from the same PEAKs database search (so they should be compatible).

Please let me know if you have any suggestions, thanks!

Cheers,
Ciaran

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Skyline
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ERROR: index 3128 out of range in mzXML file '.\C240501_C.elegans_Lib_100ng_17min_dda_141.d.mzxml'

Command-line: C:\Users\ciara\AppData\Local\Apps\2.0\7GXGT77K.ABK\AJO9DCLR.Z52\skyl..tion_9286511f3362df93_0017.0001_cdc87d1507aa7380\BlibBuild -s -A -H -v warn -o -i P -S "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant202406050814.stdin.txt" "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant.blib"
Working directory: C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_141
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OK More Info
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System.IO.IOException: ERROR: index 3128 out of range in mzXML file '.\C240501_C.elegans_Lib_100ng_17min_dda_141.d.mzxml'

Command-line: C:\Users\ciara\AppData\Local\Apps\2.0\7GXGT77K.ABK\AJO9DCLR.Z52\skyl..tion_9286511f3362df93_0017.0001_cdc87d1507aa7380\BlibBuild -s -A -H -v warn -o -i P -S "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant202406050814.stdin.txt" "C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_142\P.redundant.blib"
Working directory: C:\Users\ciara\Dropbox\KU_Leuven\1_Prohormone_convertase_project\Skyline\2024.06.05_initial_spectral_library\Peptide_library_ran_seperately\Peptide_library_141
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass, Boolean forceTempfilesCleanup, Func`3 outputAndExitCodeAreGoodFunc, Boolean updateProgressPercentage) in C:\proj\skyline_23_1\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 181
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_23_1\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 433
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 161
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Nick Shulman responded:  2024-06-05 13:25
Can you send us your files?
It looks like there were some files attached when this was first posted but I cannot see those attachments now.
You can also upload your files here:
https://skyline.ms/files.url

-- Nick
 
ciaranmc 28 responded:  2024-06-05 13:38
I have uploaded those via the link, thanks for looking at them for me.

Cheers,
Ciaran
 
Nick Shulman responded:  2024-06-05 14:34
Thank you for sending those files.
I am not sure exactly what is going on, but it is unusual to use the ".mzXML" format with this sort of data.
mzXML is a very old format, and I do not believe that it has support for things like ion mobility.

The error that you are getting "index 3128 out of range in mzXML file" is telling you that your peptide search results file is referring to a spectrum which does not exist in the mzXML file.

My guess is that the .mzXML file that you have is not the same as the file which was searched using PEAKS.

Do you happen to have a .mzML file instead?
mzML is a much more modern format than mzXML
-- Nick
 
ciaranmc 28 responded:  2024-06-06 06:02
Hey Nick,

Thanks for your response.

The .mzXML files was the output from peaks, we searched raw TimsTOF data (.d folder). PEAKS 11 does not provide a .mzML files export format, but it does allow me to export a .mgf and a refined .mgf file, as well as the .mzXML file.

Is it possible to use the .mgf files instead, or is it possible to convert any of these files to a .mzML files. The .mzML file seems to be the only format that is suitable for PEAKS analysis on MS skyline (though it is not possible to export this format from PEAKS 11, atleast from TimsTOF derived searches).

Please let me know if you know of a way for me to convert the exported files to the appropriate format for skyline.

Thanks.

Cheers,
Ciaran
 
Nick Shulman responded:  2024-06-07 16:21
Ciaran,

Yes, I believe that if you have a .mgf file you should try to use that instead of the .mzXML file that you are using.
At the end of the following support request there is an answer which says that the spectrum IDs in a PEAKS do not make any sense in .mzXML files and you should use a .mgf or .mzML file instead:
https://skyline.ms/announcements/home/support/thread.view?rowId=24811
-- Nick