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Unable to read mzML file? mnt  2022-02-05 02:04
 

Hi Skyline crew

I'm unaware if this is a known issue or not. However in an experiment of mine we are attemtpting to match a bunc of peptides to the orriginal mzML files, the opperation is partialy succesful, however in around 40% of the files it generates an error as whown below.

There were 2 failed import attempts.

At 10.56:
Failed importing results file 'D:\Data\Martin\P036\05_Skyline_IN\20211019_TimsTofOff_1ul_P036_S16_D315C25_R1_Slot2-17_1_1052.mzML'.
Object reference not set to an instance of an object.

At 10.50:
Failed importing results file 'D:\Data\Martin\P036\05_Skyline_IN\20211019_TimsTofOff_1ul_P036_S16_D315C25_R1_Slot2-17_1_1052.mzML'.
Object reference not set to an instance of an object.

What does this mean and how do i fix it?

The mzMLD files in question are generated via Proteowizard, all the files in this batch are generateed using the same settings, yet approximately 40% of the files fails.

Can anyone offer any insight into this phenomonon?

Best
Martin
 
 
mnt responded:  2022-02-05 05:40

I'm not sure if this helps, but the full error description is below:

At 14.34:
Failed importing results file 'D:\Data\Martin\P036\05_Skyline_IN2\20211019_TimsTofOff_1ul_P036_S6_D70C35_R2_Slot2-12_1_1060.mzML'.
Object reference not set to an instance of an object.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'D:\Data\Martin\P036\05_Skyline_IN2\20211019_TimsTofOff_1ul_P036_S6_D70C35_R2_Slot2-12_1_1060.mzML'.
Object reference not set to an instance of an object. ---> System.NullReferenceException: Object reference not set to an instance of an object.
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.Spectra.get_PercentComplete() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 861
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.UpdatePercentComplete() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 635
at pwiz.Skyline.Model.Results.SpectraChromDataProvider.GetChromatogram(Int32 id, Target modifiedSequence, Color peptideColor, ChromExtra& extra, TimeIntensities& timeIntensities) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\SpectraChromDataProvider.cs:line 624
at pwiz.Skyline.Model.Results.ChromData.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\ChromData.cs:line 77
at pwiz.Skyline.Model.Results.ChromDataSet.Load(ChromDataProvider provider, Target modifiedSequence, Color peptideColor) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\ChromDataSet.cs:line 295
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Load(ChromDataProvider provider) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 144
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 439
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_21_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

Best
Martin
 
Nick Shulman responded:  2022-02-05 08:31
Martin,

I have seen this error before. When I saw this error, the problem was that the retention time range over which Skyline thought it was supposed to extract chromatograms did not match the time range in the raw file. So, by the time Skyline got to the end of the raw file, not a single spectrum had been used in any chromatogram.
This problem was caused by having a spectral library where the retention times were much larger than they were supposed to be, and the workaround was to go to:
Settings > Transition Settings > Full Scan
"Retention time filtering" had been set to "Use only scans within X minutes of MS/MS IDs" and choosing a different option such as "Include all matching scans" fixed the problem.
I could also imagine that this problem might be caused by molecules having their "Explicit Retention Time" set to much too large of a number.

I should fix this bug in Skyline.
It would be helpful if we could see your Skyline document and one of your mzML files.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document.

Files which are less than 50MB can be attached to this support request.
If that .zip file and/or the .mzML file are larger than that you can upload them here:
https://skyline.ms/files.url

-- Nick
 
mnt responded:  2022-02-06 23:51
Hi Nick

Thanks a lot for the quick reply. Your solution worked perfectly, thus i did the following:
* Went to : Settings > Transition Settings > Full Scan
* Here I chose : "Include all matching scans"

Note that picking a huge time window (30 min) still did not work.

I got no clue why these files were so problematic as they looked similar (on the TIC at least) and were genereted as part of the overall batch.

For this particular project, i am unable to share any data. Sorry, also, the files are way to big for sharing (atleast 2Gb pr file, and thers a lot of files). I can tell you that the solution worked though, thus you are onto the correct track, i think.

-- Martin