Unable to Import Result Files due to multiple chromatograms

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Unable to Import Result Files due to multiple chromatograms stephanie  2020-05-28
 

Hi,

I am trying to import results (from a byonic search) for full scan filtering. I created my library and a background proteome. I have a custom labeling system with labeled R/M and methyl groups. My samples are fractionated. I have tried adding samples as multiple replicates with multiple injections, single injection, and one new replicate but I get a similar error message (see below).

I was working with three libraries and importing multiple replicates. I am going to go back and re-build my library as a single library. Not sure if that will help.

Do you have any suggestions? If there is one peptide like this, there will be more.

Best,
Stephanie

Error Message:
At 8:44 PM:
Failed importing results file 'C:\Users\slynn\Documents\3Plex_Fraction_Skyline\ADMA Fraction\20200113_SML_ADMACombo_HILIC_10.raw'.
Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.

Inner exceptions:
Exception type: System.InvalidOperationException
Error message: Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.
Unable to process chromatograms for the molecule 'YMHR[+18]NR' because one chromatogram ends at time '23.7843837738037' and the other ends at time '25.4307174682617'.
at pwiz.Skyline.Model.Results.PeptideChromDataSets.AddDataSet(ChromDataSet chromDataSet) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 838
at pwiz.Skyline.Model.Results.PeptideChromDataSets.Add(PeptideDocNode nodePep, ChromDataSet chromDataSet) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\PeptideChromData.cs:line 794
at pwiz.Skyline.Model.Results.ChromCacheBuilder.AddChromDataSet(Boolean isProcessedScans, ChromDataSet chromDataSet, PeptidePrecursorMz peptidePrecursorMz, IDictionary2 dictPeptideChromData, ICollection1 listChromData, ChromFileInfo fileInfo) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 670
at pwiz.Skyline.Model.Results.ChromCacheBuilder.CalcPeptideChromDataSets(ChromDataProvider provider, List1 listMzPrecursors, HashSet1 setInternalStandards) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 510
at pwiz.Skyline.Model.Results.ChromCacheBuilder.Read(ChromDataProvider provider) in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 371
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_20_1_x64\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 252

 
 
Nick Shulman responded:  2020-05-28
That error happens if you have two identical molecules in the Targets tree, but they have different values specified for the things that control how long of a chromatogram Skyline should extract (usually "Explicit Retention Time" or "Explicit Retention Time Window").
Usually this is a mistake-- you did not intend to have two copies of the same molecule in the Targets tree, and you can solve this by deleting the extra one.

If you would like, you can send us your Skyline document and we can figure out what is going wrong.

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 is less than 50MB you can attach it to this support request.

Otherwise you can upload it here:
https://skyline.ms/files.url

-- Nick
 
stephanie responded:  2020-05-28
I loaded a zip file for you in the file list. Your name is in the description.
Thanks for your help and the quick reply!
Stephanie
 
Nick Shulman responded:  2020-05-28
Thanks for sending us that file.

One thing that you could do to prevent this error from happening would be to go to:
Settings > Transition Settings > Full Scan
and change the "Retention Time Filtering" to "Include all matching scans".

You have two peptides in your document where the light form of the peptide is "YMHR[+18]NR", but they have different heavy forms under them:
One has:
YMHR[+18]NR
YM[+1]HR[+24.1]NR[+6]
YM[+4]HR[+28]NR
and the other has:
YMHR[+18]NR
YM[+1]HR[+24.1]NR[+6]
YMHR[+28]NR

Chromatograms are stored in the .skyd file according to the modified peptide sequence and precursor m/z, so those two TMHR[+18]NR things share a chromatogram.

When Skyline wants to look up the retention times of the MS/MS scans for a precursor, it includes the retention times of the other heavy precursors under the same peptide. Because the two peptides have different precursors under them, Skyline gets a different answer as to the set of retention times where the peptide was detected.

I will try to fix this in an upcoming release of Skyline-Daily.
-- Nick