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DIA with overlapping windows, MSFragger output files, import peptide search creates Warning: Did not find spectrum

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DIA with overlapping windows, MSFragger output files, import peptide search creates Warning: Did not find spectrum jennifer schwarz  2024-01-31 04:35
 

Dear all,

I am using overlapping windows in DIA and demultiplex the spectra with MSConvert.
The created mzML files I use for the search with FragPipe(v21.1)/MSFragger (4.0).
The created interact-*.pep.xml I use for the "import peptide search" (Skyline 23.1.0.380) .
This created the error shown in the screenshot. "could not find scan number" and "did not find spectrum".
Different score values don't make a difference.
This issue was not there in cases when I did not have overlapping windows and did not need to use MSConvert first.

All relevant files are in one folder.
I have the following files:
interact-naming_rank1.pep.xml files
naming_rank1.pepXML files
the mzML files from MSConvert used for the search
and the Thermo raw files.

The difference to the runs without prior usage of MSCovert is that those files are having the naming:
name_uncalibrated.mzML
 
 
Nick Shulman responded:  2024-01-31 14:59
I do not know what might be going on but if you send us your files we might be able to figure it out.

I am not sure exactly which files we would need in order to reproduce this problem, but you could package whatever files you think might be necessary into a .zip file and upload it here:
https://skyline.ms/files.url
-- Nick
 
jennifer schwarz responded:  2024-02-02 01:10
Dear Nick,

Thank you for taking a look at this. I have uploaded the data.
 
Nick Shulman responded:  2024-02-02 15:26

Thank you for sending that .zip file.

I can actually get things to work if I trick BiblioSpec into thinking that this pep.xml file came from XTandem instead of either Peptide Prophet or MSFragger.
That is, I took your file "interact-Zelda_240116_P3082_JS_JS_CIS3_2h_8mz_overlap_REP1_R1_rank1.pep.xml" and removed lines everything from <peptideprophet_summary on line 5 to and the point after </peptideprophet_summary> on line 7, and also removed the:
search_engine_version="MSFragger-4.0"
on line 20.
(I have attached a .zip file which contains my edited .pep.xml file)

I imagine that you could get this working on all of your files by doing these same edits to each of your interact...pep.xml files.

I will ask my coworkers and find out whether there is something we need to fix in BiblioSpec to make it so that these sorts of edits are not necessary.
-- Nick