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Spectral Library build Issue whdgka75  2024-10-01 18:54
 

We are trying to build a library using raw files of mass spectrum data contributed in other existing studies.

But as the skyline is updated, some problems arise.

During the initial file upload process, RAW format files cannot be uploaded. (picture 1)

At this stage, only ".dat" file formats are read, and ".mzid" and ".mzxml" files are not readable.

At this stage, may I know if there are any file formats that can be uploaded?

If only a specific file format is possible to uploaded, I would like to know if the library cannot be built if the contributed mass spectrum data does not have that file format.

We built the library by uploading the ".dat" file in the first step and registering the ".raw" file in the next step.

In the next step, enter “search condition”, and under “modification” I want to select carbamidomethyl(M).

However, carbamidomethyl(M) is said to already exist, but cannot be found in the list. (picture 2)

In this case, carbamidomethyl(M) is not in the list because it is a modification that is applied by default, and I would like to know if it is applied automatically or if another error occurred.
 
 
Nick Shulman responded:  2024-10-01 19:24
When you do "File > Import > Peptide Search", the first step is to give Skyline your peptide search result files.

Skyline uses a program called "BiblioSpec" to build a spectra library from peptide search results.
The complete list of peptide search result formats that BiblioSpec understands is on this page:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild
".mzid" is one of the supported peptide search result formats that you could use.

You cannot use ".mzXML" files at this first step of building a spectral library because .mzXML files do not contain any information about peptide identifications.

After you have provided the peptide search result files, BiblioSpec usually needs to find files containing spectra. These might be .raw, .mzML, .mzXML, or any of a long list of possible formats. If BiblioSpec is unable to find the spectra files, you will get an error, so it sounds like BiblioSpec probably found the .mzXML files that you were hoping to use.

I am not sure why you had an error adding the Carmidomethyl (C) modification. By the time that the wizard asks about modifications, the spectral library has already been built. It is OK to cancel out of the wizard at that point.

The Import Peptide Search wizard is roughly equivalent to:
1. Use the "Build" button on the "Library" tab at "Settings > Peptide Settings" to build a spectral library
2. Use "File > Import > FASTA" to add proteins to the document
3. Use "File > Import > Results" to tell Skyline to extract chromatograms.

Sometimes after successfully getting through the library building part of the wizard you will want to cancel the wizard and then use the "View > Spectral Libraries" menu item to see what peptides are in the library.

I hope this helps and let us know if you get stuck anywhere else.
-- Nick