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Failes on importing the data acquired with FAIMS and used CV value spectrum filter active

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Failes on importing the data acquired with FAIMS and used CV value spectrum filter active david  2023-09-27 03:16
 

Dear all,

I have some data acquired using Fusion Lumos with the FAIMS installed and two CV voltages set. When I import the file without any spectrum filter, the data are imported correctly and one can see zig-zag style of the MS1 level intensities due to the the intensities from the two CV values combined into a single graph. When I set the spectrum filter in a way to select only CV values of -50 and -70, it gives me the following error:

At 12:05 PM:
Failed importing results file 'U:\712006-Proteomics\Instruments\Orbitrap_Lumos\Data\2023\230926_IS270_Lumos1\IS270_Lumos1_74IS_FAIMS(-50-70)_HCD-OT_DDA_HeLa_50ng_01.raw'.
Times (4672) and intensities (2336) disagree in point count.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'U:\712006-Proteomics\Instruments\Orbitrap_Lumos\Data\2023\230926_IS270_Lumos1\IS270_Lumos1_74IS_FAIMS(-50-70)_HCD-OT_DDA_HeLa_50ng_01.raw'.
Times (4672) and intensities (2336) disagree in point count. ---> System.IO.InvalidDataException: Times (4672) and intensities (2336) disagree in point count.
at pwiz.Skyline.Model.Results.ChromCollector.ReleaseChromatogram(Byte[] bytesFromDisk, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCollector.cs:line 120
at pwiz.Skyline.Model.Results.ChromGroups.ReleaseChromatogram(Int32 chromatogramIndex, Single retentionTime, ChromCollector collector, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCollector.cs:line 633
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Collectors.ReleaseChromatogram(Int32 chromatogramIndex, ChromGroups chromGroups, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 1314
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, ChromatogramGroupId chromatogramGroupId, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 619
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, ChromatogramGroupId chromatogramGroupId, Color peptideColor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 84
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, ChromatogramGroupId chromatogramGroupId, Color peptideColor) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 285
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 146
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 427
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\pwiz\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 256
--- End of inner exception stack trace ---

I have tested this on the freshly opened Skyline document with just a single iRT peptide in the list of peptides to import. It does work with the positive CV values used and also with the negative CV values for which there are no data actually present in the raw file. It simply returns no traces at this case. But when I set any CV value that is in the data (-50 or -70), I get the abovementioned error.

I have used 23.1.1.268 Skyline version.

Let me know if you would need any more data, I could share the raw file if necessary.

Thank you for double checking!

David
 
 
Nick Shulman responded:  2023-09-27 05:42
I think this error must be caused by a bug in Skyline.

Can you send us your Skyline document and your .raw file?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file and your .raw file are less than 50MB you can attach them to this support request.
You can upload larger files here:
https://skyline.ms/files.url

-- Nick
 
david responded:  2023-09-27 06:23
Thank you for a quick response.

Here is the link to get the zip file got if that is not a problem for you: https://filesender.cesnet.cz/?s=download&token=81eec167-5d5a-4274-ab1d-a173633d2376

I have removed the spectrum filter in the shared document to enable the raw data import. So now there is one file imported successfully but no filter set. You will need to set the spectrum filter to filter for CV values of -50 or -70 to get the error I have seen; at least that is the way it behaves on my system.

Thank you again,
David
 
Nick Shulman responded:  2023-09-27 07:18
Thank you for sending those files. I will try to fix this bug soon.

There is a workaround that you could do until I manage to fix this bug.
If you go to "Settings > Transition Settings > Full Scan" and change the "Retention time filtering" to "Use only scans within X minutes of MS/MS IDs" then Skyline will not run into this bug. You could make that "X" number very large such as 100 so that it won't actually have any effect on the chromatogram that gets extracted.

The problem is that there is a bug in the new "Spectrum Filter" feature, and, in this case, Skyline does not realize that the MS1 chromatograms for your different precursors are going to have a different number of points in them. If you change the "Retention time filtering" setting at "Settings > Transition Settings > Full Scan", then Skyline will not make this assumption and you will be able to successfully extract chromatograms without error.

Thank you for your feedback on the Spectrum Filter feature. This is a new feature in Skyline 23.1, and I see that it still has a few bugs that need to be worked out.
-- Nick
 
david responded:  2023-09-27 07:56
Thank you Nick for a quick check and the workaround! I have tested it now and works as expected, thx a lot! The spectrum filter is killer feature for me at least so it is great it could be used with the workaround.

Thank you once more, happy to help.

Take care,
David