Unable to finish importing chromatograms because the retention time predictor linear regression failed.

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Unable to finish importing chromatograms because the retention time predictor linear regression failed. paga  2021-11-04
 
Good evening,

Ive been struggling with a diaPASEF (Bruker) analysis on Skyline. The experiment's goal is to find a set of 30 heavy labeled peptides on one DIA-file. I've produced a Skyline library using PEAKS but after loading the single DIA file it keeps fails. Ive been guiding the setting parameters based on the "Analysis of diaPASEF data in Skyline" document but including precursors as it is immunopeptidomics.

When specifying the use of Biognosys C-11 iRT peptides, I got the following error.

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Skyline version: 21.1.0.278-fe50e66f2 (64-bit)
Installation ID: 5bc1d5e0-0694-49c2-87b5-7e376a14cafe
Exception type: InvalidDataException
Error message: Found invalid data while decoding.

System.IO.InvalidDataException: Found invalid data while decoding.
   at pwiz.Skyline.Model.ImportPeptideSearchManager.LoadBackground(IDocumentContainer container, SrmDocument document, SrmDocument docCurrent) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\ImportPeptideSearch.cs:line 602
   at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
Exception caught at:
   at pwiz.Skyline.Model.BackgroundLoader.OnLoadBackground(IDocumentContainer container, SrmDocument document) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\BackgroundLoader.cs:line 130
   at System.Threading.ExecutionContext.RunInternal(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state, Boolean preserveSyncCtx)
   at System.Threading.ExecutionContext.Run(ExecutionContext executionContext, ContextCallback callback, Object state)
   at System.Threading.ThreadHelper.ThreadStart()
--------------------

After trying with the "Automatic" determination of iRT peptides, I got the following error


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Failed importing results file 'D:\DIA-PASEF_optimisation_2021\202109_Sr_DIAPSEF_EVAXheavy_RawData\10-7-2021_DIA_100ms_Im1.7_Evax_hela400_2sec_22-01-15_Slot1-10_3097.d'.
The calculator From_scratch_Automatic-iRT requires all of its standard peptides in order to determine a regression.
--------------------
 I will attached some of these file after the failed attempt.
 
 
Nick Shulman responded:  2021-11-04
If you want to send us your Skyline document you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including iRT database and extracted chromatogram.

If that .zip file is less than 50MB you can attach it to this support request.
You can upload larger files here:
https://skyline.ms/files.url

The first error that you ran into happened because by the time Skyline finished extracting chromatograms there were no longer any decoy peptides in the document. I believe this can happen if you delete those peptides from the document in between the time when you start doing the Import Peptide Search and when it gets all the way to the end.

The second error is happening because you do not have the full set of iRT standard peptides in your document. You can see the list of iRT standard peptides if you go to:
Settings > Peptide Settings > Prediction > Calculator Button > Edit Current

If you want Skyline to add the iRT standard peptides to your document again you can go to:
Settings > Peptide Settings > Prediction
and change "Retention time predictor" to "None".
Then press OK on the Peptide Settings dialog and then again go to:
Settings > Peptide Settings > Prediction
and change "Retention time predictor" back to its original value.

When you press "OK" on the Peptide Settings dialog a second time Skyline will offer to add the missing iRT standard peptides to your document.

-- Nick
 
paga responded:  2021-11-04
Thank you Nick, I managed to upload the files under the names: With_Biognosys_iRT.zip and With_Automatic_iRT.zip
 
Nick Shulman responded:  2021-11-05
Thanks for sending those files.

I am not sure what exactly you need help with.

If your Skyline document has an iRT predictor, then, when you extract chromatograms, Skyline first extracts chromatograms for the iRT standards, finds the chromatogram peaks, and then does a linear regression between where those peaks were found and the iRT score for those peptides. If that line is not straight enough, then you get an error:
Unable to finish importing chromatograms because the retention time predictor linear regression failed.

When you get that error, you end up only having chromatograms for the iRT standards, and all of the other peptides in your document will be missing chromatograms.

What you need to do then is go to:
View > Retention Times > Regression > Score to Run
and then you have to right-click on the linear regression graph and choose the following three things:
Calculator > From_scratch (that's the name of your retention time predictor at "Settings > Peptide Settings > Prediction > Calculator Button"
Points > Standards
Set Threshold > 0.99

After you have done all that, Skyline will show you a graph where the purple points are the ones where peptide was found in a location which does not match where its iRT value says it should have been.

In your case, it looks like most of your standards were not found where they were supposed to be. I do not know much about how to use a mass spectrometer so I am not sure what might be going wrong. The iRT standards are supposed to have been spiked into the sample at sufficient concentration to be easy to detect.
-- Nick