Dear Skyline support,
I have managed to do this before, but now I am again running into issues when importing MyriMatch search results into Skyline. I am trying to import these peptide search results to build a library in the "peptide settings -> library -> build" menu. However, Skyline does not recognize the files (MyriMatch results parsed through Peptide Prophet - this has, however, worked before). It would be great if you could please have a look at the attached example file here and let me know what is wrong with it or why Skyline can't load it.
In general, we have previously managed to use .mzXML and files like the attached one to build libraries with the DDA with MS1 filtering workflow.
It is my understanding that I load the raw files (.mzXML) using "Import Peptide Search, build spectral library, start from DDA raw (search and build library)" and the peptide search results (pep.XML) under "peptide settings -> library -> build"? Is this correct or am I completely off?
Skyline recognizes both the .mzXML files as well as thermo .raw files when I use the "Import Peptide Search, build spectral library, start from DDA raw (search and build library)" but after the percolator it always says "Search failed". Do you know why that is? I attach the corrsponding mzXML file, too.
I would really appreciate your help on this!
Many thanks,
Claudia
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| Nick Shulman responded: |
2024-03-16 22:17 |
It sounds like you are saying that you attached a file to this support request, but I do not see any attachments.
That often happens if the files you were trying to attach were larger than the limit which is 50MB.
You can upload larger files here:
https://skyline.ms/files.url
--Nick |
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| staab-weijnitz responded: |
2024-03-18 21:13 |
Hi Nick,
I uploaded some representative files that I try to build the library from in a folder called "ForSkylineforum"; I later realized that I should better call it something more descriptive and then made a folder called "Staab-Weijnitz_for_Skyline". [Sorry about the confusion, I was doing this today during and between meetings and constantly got error messages during the upload, so I thought none of the uploads had been successful.]
Both folders contain almost the same, "ForSkylineForum" contains the Fasta file in addition.
For building the library, I got Skyline in the end to use the interact... .pep.xml files, the .mzXML (converted from thermo .raw files by MS Convert, TPP) and the fasta file. This seemed to work, at least finally Skyline recognizes the pep.XML files! But even though in ID picker I see many many different proteins including the collagens I am interested in, Skyline does not match anything to proteins in the fasta file and I get the message: "Importing the FASTA did not create any target proteins".
Many thanks in advance,
Claudia |
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| Nick Shulman responded: |
2024-03-19 03:05 |
I am not seeing anything like the "Importing the FASTA did not create any target proteins" message.
Here is the Skyline document that I ended up with after doing "Import > Peptide Search" with your files:
https://skyline.ms/_webdav/home/support/file%20sharing/%40files/nicksh_staab_weignitz_result.sky.zip
Can you send me the Skyline document that you ended up with when you saw that message? If you were going through the "File > Import > Peptide Search" wizard, you can press the "cancel" button at whatever stage of the wizard you were at when you saw that message.
Then you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including the spectral library that you might have created.
If you send me that .zip file, I might be able to figure out what is going wrong for you.
-- Nick |
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| staab-weijnitz responded: |
2024-03-19 11:36 |
Dear Nick,
Well, your Skyline doc looks fine!
Many thanks, I attach the .zip file of mine. A .skyd file was not among the output files I got.
Best,
Claudia |
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| staab-weijnitz responded: |
2024-03-19 11:42 |
Dear Nick,
Well, your Skyline doc looks fine!
Many thanks, I uploaded the .zip file of mine into the same folder (ForSkylineForum). A .skyd file was not among the output files I got.
Best,
Claudia |
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| Nick Shulman responded: |
2024-03-19 13:15 |
Thank you for uploading that .zip file.
I was hoping that I would be able to do "File > Import > FASTA" and then see the message that you were seeing about no proteins being added.
However, I end up seeing plenty of proteins when I do that, so I do not know why you might have been having a problem before.
Let me know if there is anything else we can help you with.
-- Nick |
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| staab-weijnitz responded: |
2024-03-19 16:15 |
Hmm, when I tried to build the library via the -> Import -> Peptide Search menu using the same FASTA file it did not work.
However, now, after having imported the FASTA file like you described, adjusting the transition settings, and then importing Results, that worked for 8 out of 20 files. So, that's already a major improvement! Thank you so much! :)
One issue remains: There are several files that will not load and I get the errors below. Any idea what might be the problem here?
Thanks,
Claudia
________________
Short version:
Failed importing results file 'C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML'.
[ReaderFail] don't know how to read C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML
Long version:
Failed importing results file 'C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML'.
[ReaderFail] don't know how to read C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML
pwiz.Skyline.Model.Results.ChromCacheBuildException: Failed importing results file 'C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML'.
[ReaderFail] don't know how to read C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML ---> System.Exception: [ReaderFail] don't know how to read C:\Users\staabwec\OneDrive - The University of Colorado Denver\Documents\Data\FKBP10kd\FKBP10_PTManalysis\MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML
at pwiz.CLI.msdata.ReaderList.read(String filename, MSData result, Int32 runIndex, ReaderConfig config)
at pwiz.ProteowizardWrapper.MsDataFileImpl..ctor(String path, Int32 sampleIndex, LockMassParameters lockmassParameters, Boolean simAsSpectra, Boolean srmAsSpectra, Boolean acceptZeroLengthSpectra, Boolean requireVendorCentroidedMS1, Boolean requireVendorCentroidedMS2, Boolean ignoreZeroIntensityPoints, Int32 preferOnlyMsLevel, Boolean combineIonMobilitySpectra, Boolean trimNativeId) in C:\proj\skyline_23_1\pwiz_tools\Shared\ProteowizardWrapper\MsDataFileImpl.cs:line 201
at pwiz.Skyline.Model.Results.MsDataFilePath.OpenMsDataFile(Boolean simAsSpectra, Boolean preferOnlyMs1, Boolean centroidMs1, Boolean centroidMs2, Boolean ignoreZeroIntensityPoints) in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Results\MsDataFilePath.cs:line 295
at pwiz.Skyline.Model.Results.ChromCacheBuilder.BuildCache() in C:\proj\skyline_23_1\pwiz_tools\Skyline\Model\Results\ChromCacheBuilder.cs:line 191
--- End of inner exception stack trace --- |
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| Nick Shulman responded: |
2024-03-19 16:50 |
That error is saying that the data inside the file "MEJ9538A13MA3_40_341946_E26_3_MLT18_10kd_woTgfb.mzXML" is not what's supposed to be inside of a mzXML file.
If you send me that file I could tell you more about what is wrong. The data in the file might have become corrupted or truncated when it was copied from somewhere.
By the way, it's usually better to use .mzML files instead of .mzXML files.
mzXML is a very old format and does not support a lot of attributes. If you are working with tools that are able to handle both mzML and mzXML things usually work a little better with mzML.
MSConvert knows how to create both mzML and mzXML files.
-- Nick |
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| staab-weijnitz responded: |
2024-03-20 09:31 |
Thanks Nick,
Indeed the data must have been corrupted by copying, because when I loaded it from the original folder where the output from MS Convert had been originally saved, the remaining two files loaded just fine.
So - tata - I have a complete Skyline file and can start quantifying!
Thanks for your putting me on the right track with everything, much appreciated.
Best,
Claudia |
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