Unable to use MSFragger for spectral library

Unable to use MSFragger for spectral library s jager  2023-07-31 06:52


I have been using MSFragger to analyze my timstof data, and i have been trying to upload the ‘interact.pep.xml’ files as spectral library. I put the '_uncalibrated.mzML' in the parent folder as had been described previously. However, i get the following error message:

"interact.pep.xml: To import an MSFragger search of timsTOF data (with ion_mobility attribute), the corresponding *_uncalibrated.mgf or *_calibrated.mgf file is required"

As of version 3.6, MSFragger does not output these files, and does the mxML counterparts instead (which I have). Can I somehow solve this and import the spectral library.

Or is there a workaround, for example a way i can import a list of peptide sequences with modifications, and that skyline processes this similarly to when I upload the FASTA?

thanks in advance!

Nick Shulman responded:  2023-08-02 14:18
I believe that the reason that BiblioSpec requires the .mgf files with timstof data is that it is impossible to figure out which spectrum in the .mzML file corresponds to the spectrum numbers in the pep.xml file.

If you send us all of your MSFragger search results, we might be able to figure out some sort of workaround.
You can package everything up in a .zip file and upload it here:

To answer your other question, you can use the Skyline menu item:
Edit > Insert > Peptides
to insert peptides with modifications.

Before you do that, you should go to:
Settings > Peptide Settings > Modifications
and tell Skyline about all of the modifications that you are going to be using.

In the peptide sequences in the "Edit > Insert > Peptides" window, you can either specify a modification as a mass offset (e.g. "SEQUENC[+57.02]EK") or using the name of the modification (e.g. "SEQUENC[Carbamidomethyl (C)]EK").
-- Nick
s jager responded:  2023-08-10 04:17
Hi Nick,

Thank you for your reply! I have uploaded the zip file named: "TimsTOF_MSFragger.zip".
Nick Shulman responded:  2023-08-10 08:35
Thank you for sending those search results.
Can you also send me the inputs to those search results? That might be the .mzML, .mgf, or maybe .d folders.
I realize that will probably end up being a very big .zip file. Unfortunately, I do not know exactly which file(s) I would need, but I was hoping that if I saw all the files that you have I might be able to figure out a workaround.
-- Nick
s jager responded:  2023-08-21 05:36
Hi Nick,

I uploaded all files i could find. They are named: "TimsTOF_rawdata.zip" and they include the .d the .mzBIN and uncalibrated.mzML
Nick Shulman responded:  2023-08-21 16:31
Thank you for sending those raw files.

I think everything will work for you if you use Skyline-daily instead of Skyline 22.2.
You can install Skyline-daily by pushing the "Skyline-daily (beta)" button on the main Skyline page:

We are going to be releasing Skyline 23.1 soon, and when that happens, all of the features and fixes that are currently in Skyline-daily will be present in Skyline 23.1.

If you copy the "*uncalibrated.mzML" files from the .zip file that you uploaded, and put them in the "output" folder from the other group of files that you gave me, then BiblioSpec find those mzML files when building a spectral library from your "interace.pep.xml" files.

When BiblioSpec is looking for the .mzML file to go with a pep.xml file, I believe it looks in the same folder as the pep.xml as well as the parent and grandparent folders which is why putting those .mzML files in the "output" folder works with this dataset.
-- Nick