"Failed importing results...The calculator ... requires all of its standared peptides in order to determine a regression

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"Failed importing results...The calculator ... requires all of its standared peptides in order to determine a regression Mark Athanason  2020-10-05
 

Hello,

I'm getting the following error, seen below. I'm simply trying to build a spectral library to perform DIA quant with 2 DDA runs. I've never seen this error before. I'm using the most current version of skyline daily. For RT standards, I selected automatic because when I select biognosys 10 or 11 it doesn't find any standards, for some reason. When selecting automatic, it finds 10 standards with a r^2 = 1. Going down the wizard is fine, then when it's importing the DDA data it cancels and throws this error:

At 11:25 AM:
Failed importing results file 'E:\Mark\spheroid\firstPlate\DMSO_DDA_1.mzXML'.
The calculator DMSO1 requires all of its standard peptides in order to determine a regression.
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'E:\Mark\spheroid\firstPlate\DMSO_DDA_1.mzXML'.
The calculator DMSO1 requires all of its standard peptides in order to determine a regression. ---> pwiz.Skyline.Model.Irt.IncompleteStandardException: The calculator DMSO1 requires all of its standard peptides in order to determine a regression.
at pwiz.Skyline.Model.Irt.RCalcIrt.ChooseRegressionPeptides(IEnumerable`1 peptides, Int32& minCount) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Irt\RCalcIrt.cs:line 152
at pwiz.Skyline.Model.Results.ChromCacheBuilder.RetentionTimePredictor.CreateConversion() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 838
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 404
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_2_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252
--- End of inner exception stack trace ---

The search was performed with comet with .RAW files converted to .mzXML with default settings. The output file is a pep.xml. I think the error is occurring when it's trying to automatically train the mProphet model. In the search settings I performed a concatenated decoy search, with skyline being told to make 1 reverse decoy to train mProphet.
Any help would be appreciated,
Mark

 
 
Kaipo responded:  2020-10-05

Hi Mark,
Would you mind uploading the mzXML/pep.xml files to the file share? https://skyline.ms/files.url

Thanks,
Kaipo

 
Nick Shulman responded:  2020-10-05
Can you send us your Skyline document?

In Skyline, 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 chromatograms.

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

It would probably also be helpful if you could send us that file "DMSO_DDA_1.mzXML".

When Skyline is extracting chromatograms, and you have an iRT predictor, Skyline first extracts chromatograms for the iRT standards. Skyline finds the peaks for those iRT standards, and then does a linear regression in order to find the slope and intercept to convert between an iRT score (which is typically a number between 0 and 100) and the predicted retention time.

If anything goes wrong with that step of doing the linear regression, then you are usually left in a state where only your iRT standards have chromatograms for that replicate, but all of the other peptides in your document have no chromatograms for that replicate. Then, you are supposed to go to:
View > Retention Times > Score to Run
and see if you can tell what went wrong.

In your case, though, the error is saying that your document is missing some of the iRT standard peptides. That is, they have been deleted from the Targets tree, or they were never there. Since you said that Skyline automatically chose the standards for you, I would have expected them to at least have been present when Skyline chose the standards.

It will probably be easier for us to figure out what is going wrong when we see your Skyline document.
-- Nick