Frustrated with Library Build jcarey  2021-11-19

Dear Skyline Support Team,

I have watched many of the webinars and completed most all of the tutorials successfully and still cannot build a library from MS data from a commercially available Shingrix vaccine. Data were aquired on a Waters QTOF. I've checked many of the library build Support threads and tried to follow the steps suggested by Skyline Support Staff, but still only get back error messages related to invalid file type.

As a last resort today, the data was redownloaded from our vendor and kept in the download folder for simplicity. The library Build function was used and the destination for the "to be created" .blib file was also chosen to be the exact same location in the download folder (have tried many permutations of file locations to no avail). The source file was specified using the Build dialog box and when the "Next" button was clicked the error message below was received. I have tried many, many ways to load MS results as a library and consulted lots of references. Seems like I need some hand-holding at this point. Thanks!

System.IO.IOException: ERROR: Error with '_FUNC001.DAT'. Invalid results (.dat) file.

Command-line: C:\Users\Jim\AppData\Local\Apps\2.0\JA9V9166.9KW\61QRGA69.CNO\skyl..tion_e4141a2a22107248_0015.0001_62a9d240a069a6b4\BlibBuild -s -A -H -o -c 0.95 -i Shingrix_19NOV21_blib -S "C:\Users\Jim\AppData\Local\Temp\tmpFE0A.tmp" "C:\Users\Jim\Downloads\Shingrix_19NOV21_blib.redundant.blib"
Working directory: C:\Users\Jim\Downloads\MC_20210909_ADES1407_Shingrix.raw
at pwiz.Common.SystemUtil.ProcessRunner.Run(ProcessStartInfo psi, String stdin, IProgressMonitor progress, IProgressStatus& status, TextWriter writer, ProcessPriorityClass priorityClass) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\Common\SystemUtil\ProcessRunner.cs:line 142
at pwiz.BiblioSpec.BlibBuild.BuildLibrary(LibraryBuildAction libraryBuildAction, IProgressMonitor progressMonitor, IProgressStatus& status, String& commandArgs, String& messageLog, String[]& ambiguous) in C:\proj\skyline_21_1_x64\pwiz_tools\Shared\BiblioSpec\BlibBuild.cs:line 201
at pwiz.Skyline.Model.Lib.BiblioSpecLiteBuilder.BuildLibrary(IProgressMonitor progress) in C:\proj\skyline_21_1_x64\pwiz_tools\Skyline\Model\Lib\BiblioSpecLiteBuilder.cs:line 157

Nick Shulman responded:  2021-11-19
Spectral libraries are usually built from peptide search results.
It sound like you are trying to build a spectral library from Waters raw mass spectrometry data, which is not going to work.

One thing that you could do is ask Skyline to perform a peptide search on your raw data. To do that, you would go to:
File > Import > Peptide Search
and choose "DDA Raw" in the "Start From" dropdown box on the first page of the Import Peptide Search wizard.
The wizard will guide you through the steps of selecting raw files, and providing a protein FASTA file.

By the way, this web page lists all of the different types of peptide search results that you can build a spectral library from:
Do you have any peptide search results in any of the formats listed on that page?

If you send us your data files we can tell you what sort of data you have, and what sorts of things Skyline can do with it.
If you would like, you can zip up your data files and upload them here:
Or, if you downloaded your data from a publicly available place, let us know where that was, and we can take a look at it there.

-- Nick
jcarey responded:  2021-11-19
Thanks so much for the quick response, Nick. I tried the File > Import > Peptide Search approach but also received an error message. The raw data file folder has been uploaded as suggested to The file name is "MC_20210909_ADES1407_Shingrix.raw" and it has my name next to it. Looking forward to learning how to use the awesome software!

Best regards,

Nick Shulman responded:  2021-11-19
Thanks for uploading your folder "MC_20210909_ADES1407_Shingrix.raw".

I am not sure what type of experiment you did. I see that you have 553 MS1 spectra that were collected over an experiment run which was 10 minutes long.
There are also 31 MS2 spectra, and every one of those MS2 spectra isolated the precursor m/z 2275.

If I had to guess what sort of experiment this was, I would say that this is a PRM experiment where the mass spectrometer was told to monitor a molecule whose m/z was 2275. That would be unusual for a PRM experiment, since usually PRM experiments target multiple molecules and end up having more MS2 spectra than MS1 spectra.

This is the PRM tutorial, which you might find helpful:

Do you know what the mass spectrometer was looking for whose m/z is 2275? The first step for analyzing this data in Skyline would probably be to tell Skyline which peptide or molecule you were looking for. You would do that with either the menu item:
Edit > Insert Peptides
Edit > Insert Transition List

You should also go to:
Settings > Transition Settings > Full Scan
and change MS1 Filtering Isotope Peaks Included to perhaps "Count"
and change MS/MS Filtering Acquisition Method to "Targeted"

after you have told Skyline which peptide or molecule to look for, you can tell Skyline to extract chromatograms with the menu item:
File > Import > Results

-- Nick
jcarey responded:  2021-11-19
Hi Nick,

This was an intact mass experiment of a commercially available vaccine that contains glycoprotein E (gE). I suspect that the MS2 signal may be a calibrant molecule, but will check with our vendor to learn more details. Maybe I'm trying to use Skyline in a way that it is not built to address. I was hoping to import the intact mass data and compare to the FASTA that is published and analyze for PTMs or missing peptides, etc. Is this a reasonable approach that falls within Skyline's capabilities?

Thanks again,

Nick Shulman responded:  2021-11-19
It sounds like you want to do quantification on MS1 chromatograms.

I would recommend the MS1 tutorial:

Also, I am not sure whether you are interested in looking at peptides or small molecules.
If you are interested in small molecules, you should take a look at the small molecule tutorial:

-- Nick
jcarey responded:  2021-11-19
Thanks, Nick.

We are ultimately looking to do intact mass on gE, MW ~ 58 kDa, and peptide mapping as a quality control for the over 500 peptides present in the protein. The work is being done by a CDMO so I will work more closely with them to see how they are acquiring the data.

We also have a small molecule that requires MS analysis for mass identification of impurities. It's MW is 1137 Da. Thank you for sending the link for that MS1 tutorial. It is different from the MS1 tutorials I've been going through. Looks to possibly be an updated version and seems to address more of our current needs. I will complete this tutorial tonight.

I believe Skyline will be a good tool for us so your help is greatly appreciated. The TIC from our small molecule MS raw data has been imported and visualized within Skyline. Perhaps with the new MS1 tutorial the MS data will be successfully imported and analyzed as well.

Thanks again for the help!