IM data in Skyline?

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IM data in Skyline? mnt  2021-10-18
 
Hello Skyline

Lately i have been attempting to analyze som data from our TimsTof Pro2 with the PASEF engaged. The workflow i'm attempting to use is the following: Data is aquiered via our TimsTof Pro 2 with PASEF engaged, the .d files are converted both to .mgf and .mzML vi MSConvert. Here the "Combine ion mobility scans" is tjecked. The .mgf file are analyzed via xiSEARCH where a .ssl file is generated for Skyline. The .ssl file is placed in a folder with the .mzML files wherefrom it is opened in Skyline as part of building my library. However, this process (buildign the library) fails with the following error:

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Skyline
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ERROR: No spectra were found for the new library.

Command-line: C:\Users\D42881\AppData\Local\Apps\2.0\QJTKV4CR.ROX\EEB2DNKN.1HP\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i TimsOnTest -K -S "C:\Users\D42881\AppData\Local\Temp\tmpDACB.tmp" "D:\MNT\Data\P035\Skyline\TimsOnTest.redundant.blib"
Working directory: D:\MNT\Data\P035\Data\mzML
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OK More Info
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System.IO.IOException: ERROR: No spectra were found for the new library.

Command-line: C:\Users\D42881\AppData\Local\Apps\2.0\QJTKV4CR.ROX\EEB2DNKN.1HP\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i TimsOnTest -K -S "C:\Users\D42881\AppData\Local\Temp\tmpDACB.tmp" "D:\MNT\Data\P035\Skyline\TimsOnTest.redundant.blib"
Working directory: D:\MNT\Data\P035\Data\mzML
   at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 142
   at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
   at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157
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The interesting thing is that this workflow works perfectly when the PASEF is not engaged. So, whatever this is, it has to do with the PASEF.
 
 
Nick Shulman responded:  2021-10-18
That error ("no spectra were found for the new library") means that BiblioSpec was not able to find spectra with in the raw (or mzML) file which correspond to the spectrum identifiers in the peptide search results files.
Sometimes this means that the format of the identifier that you are using is not what BiblioSpec is expecting.

When you combine ion mobility spectra, multiple spectra in the raw file which all have the same retention time but different ion mobility values are represented by a single spectrum in the mzML file. For this reason the spectrum identifiers that you should use for spectra in the mzML file will probably be different from what they would be in the raw file.

I am not very familiar with how you are supposed to refer to spectra in a ssl file. I know sometimes you can just use an integer representing the position of the spectrum in the list of all spectra (I can't remember whether that numbering starts at 0 or 1). For other types of raw files it is necessary to use a more complicated spectrum identifier that has some dots in it.

If you send us your files we can take a look and figure out what needs to be done.
Here is the web page which specifies what types of files you need to have depending on what type of search results you have:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild

You can zip up your files and upload them here:
https://skyline.ms/files.url

-- Nick