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ID retention times incorrect jdemeter  2025-06-19 19:56
 

Hi,

I am trying to load a dataset into Skyline-daily using fragpipe and noticed that the retention times for psms that are loaded don't agree with the retention times that are in the spectral library.

I am analyzing a Bruker timstof dda dataset. I attached the fragpipe log-file that shows how - in the last step - it is loading the result into skyline-daily. I can send you the folder with the actual data.

I also attached an excel file with an example of a peptide retention times (what is loaded and what is in the spectral library).

Can you please take a look?

Thanks,
Janos
 
 
Nick Shulman responded:  2025-06-19 20:26
Yes, please send us the folder with the actual data.
We will try building a .blib file from that data and try to figure out what is going on.
You can zip all of your files up and upload them here:
https://skyline.ms/files.url

Also, it would also be helpful if you could send us your Skyline document.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries.
-- Nick
 
jdemeter responded:  2025-06-26 15:56
Hi Nick,

Any success finding out what is causing this issue?

Thanks,
Janos
 
Nick Shulman responded:  2025-06-26 17:45
Thank you for uploading that .zip file.
I see that there is a psm.ssl file in there, but I cannot find the peptide VGAGAPVYMAAVLEYLTAEILELAGNAARDNKK in that file.
I see the peptide VGAGAPVYMAAVLEYLTAEILELAGNAARDNKK in one of the .blib files, so I think that .blib file must have been built from a different psm.ssl file.
Do you have a psm.ssl file which contains the peptide "VGAGAPVYMAAVLEYLTAEILELAGNAARDNKK" that you could send me?
In general, I would expect the retention times in the .blib file to exactly match what is in the psm.ssl file that the library was built from.
-- Nick
 
jdemeter responded:  2025-06-28 09:22
Hi Nick,

Yes, sorry about that. I uploaded a file (jdemeter_ssl_blib_rettimes.tsv) that contains a table with the retention times in ssl and redundant blib files. About 2/3 of them are different.

Thank you again for looking into this.

Janos
 
jdemeter responded:  2025-06-28 09:45
One more thing - a scatter plot of rt.blib - rt.ssl colored by file, shows an interesting pattern that might help.

Thanks,
Janos
 
Nick Shulman responded:  2025-06-28 09:48

I think I need a file named "psm.ssl" in order to figure out how the .blib file got the way that it did.
BiblioSpec spectral libraries (.blib files) are SQLite databases.
One of the tables in that database is "SpectrumSourceFiles" which lists the name of the file that the spectra came from (e.g. 250617_MS271_2-3_Slot1-18_1_5073_uncalibrated.mzML) and the peptide spectrum match file, which in this case is called "psm.ssl".

Here is what that SpectrumSourceFiles table looks like:

id fileName idFileName cutoffScore
1 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_2-3_Slot1-18_1_5073_uncalibrated.mzML psm.ssl 0.95
2 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_2-5_Slot1-19_1_5074_uncalibrated.mzML psm.ssl 0.95
3 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_GdHCl-AMG-1_Slot1-12_1_5061_uncalibrated.mzML psm.ssl 0.95
4 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_GdHCl-AMG-2_Slot1-13_1_5062_uncalibrated.mzML psm.ssl 0.95
5 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_GdHCl-DMSO-1_Slot1-10_1_5057_uncalibrated.mzML psm.ssl 0.95
6 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_GdHCl-DMSO-2_Slot1-11_1_5058_uncalibrated.mzML psm.ssl 0.95
7 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_Urea-AMG-1_Slot1-16_1_5069_uncalibrated.mzML psm.ssl 0.95
8 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_Urea-AMG-2_Slot1-17_1_5070_uncalibrated.mzML psm.ssl 0.95
9 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_Urea-DMSO-1_Slot1-14_1_5065_uncalibrated.mzML psm.ssl 0.95
10 Z:\jacksondata_2025\Ran_2025\250617_Dan\250617_MS271_Urea-DMSO-2_Slot1-15_1_5066_uncalibrated.mzML psm.ssl 0.95

If you send me "psm.ssl" as well as those .mzML files, I can try building the spectral library and see what the retention times end up being.

My guess is that the retention times in the psm.ssl file were already incorrect, and the problem came from how the psm.ssl file was generated.

I am not sure exactly how Fragpipe creates the psm.ssl file, but I think this problem is probably coming from something in the process before Skyline gets involved.
-- Nick
 
jdemeter responded:  2025-06-28 10:46

Hi Nick,

The psm.ssl file is in the zip file I uploaded previously and the file I just uploaded today has the retention times from both blib and ssl files.

Is it possible you send me an email and I give access to the mzML files for that email?

Thanks,
Janos
 
Nick Shulman responded:  2025-06-29 15:20

Thank you for sending me those files.
There is a bug in the SSL reader in BiblioSpec. The "retention-time" column should not be used if the .ssl file contains data for more than one mass spec data file.
BiblioSpec remembers what retention time was specified for each scan number, but it does not keep track of the file name.
What that means is that even though there is the following line in the .ssl file specifying that the retention time should be 4.0797:

file scan charge sequence score-type score retention-time ion-mobility ion-mobility-units
20250609_AMG1_Slot1-31_1_5024_uncalibrated.mzML 03439 2 EEEDKDDEEKPK PERCOLATOR QVALUE 0.017199993 4.0797234 0.9993 1/K0

the retention time that BiblioSpec ends up using is the one from this other line much later in the file which specifies the retention time should be 4.77. This other line has the same scan number (03439) but is for a completely different file:

file scan charge sequence score-type score retention-time ion-mobility ion-mobility-units
20250609_MEKi2_Slot1-44_1_5051_uncalibrated.mzML 03439 2 M[16.0]LPDKDC[57.0]R PERCOLATOR QVALUE 0.25590003 4.77244 0.8177 1/K0

For now, the easiest thing to do would be to remove the retention-time column from the .ssl file. When no retention time is specified, BiblioSpec will use the retention time associated with the spectrum itself.

A different workaround would be to split your .ssl files up so that each .ssl file only contains information for a single mass spec data file.

I will try to find someone to fix this bug.
-- Nick
 
jdemeter responded:  2025-06-30 19:52

Hi NIck,

Thank you for figuring out what's going on. How soon do you expect this can be fixed?

Thanks,
Janos
 
Nick Shulman responded:  2025-06-30 20:19
This will probably be fixed in Skyline-daily in a week or two.
We are working on a fix for this here:
https://github.com/ProteoWizard/pwiz/pull/3530

In the meantime I would recommend removing the "retention-time" columns from your .ssl files because it is redundant.
--Nick