Failed to execute script 'pParse2Plus' due to unhandled exception: KeyError in mgf_file.write_mgf_from_ms2_mp

[MichelDierckx]

Hello,

I am analyzing the publicly available cross-linking dataset PXD042173:
https://www.ebi.ac.uk/pride/archive/projects/PXD042173

When processing the data with pLink3, I repeatedly encounter an error originating from the pParse2Plus step. The issue occurs both when running through the GUI and via the command line.

[pParse2Plus] 0, Write mgf [ERROR] Traceback (most recent call last): File "pParse2Plus.py", line 96, in <module> File "pParse2Plus.py", line 79, in _main File "flow.py", line 386, in flow.arg_plink_cfg File "flow.py", line 339, in flow.flow_pparse2plus_cfg File "flow.py", line 300, in flow.flow_raw_cfg File "mgf_file.py", line 45, in mgf_file.write_mgf_from_ms2_mp File "mgf_file.py", line 55, in mgf_file.write_mgf_from_ms2_mp KeyError: 3324 [12872] Failed to execute script 'pParse2Plus' due to unhandled exception!

I have isolated the RAW files that reproduce the problem. One example of a problematic RAW file:
ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/08/PXD042173/L1_20211004_MxR_secondbatch_DSSOplate_B12.raw

For this individual file, the search (multi-thread, single CPU) completes despite the pParse2Plus error. However, when processing the full dataset (Multiprocess, CPU number = 16), the issue affects a substantial fraction of the files (66 out of 178), and in previous runs the GUI crashed before completion.

Any insight would be greatly appreciated.

[pParse2Plus] "C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\failed_file_example_gui\failed_file_example_gui.plink" " -t -0.8 -z 5 -i 1 -C 1 -a 0 -p 1 -S 1" " -path:pParse2 ..\pParse.exe -Plus:pxtract_n_isotope 2"
[pParse2Plus] 0, cfg_plus_args: {'path': {'pParse2': '..\pParse.exe'}, 'Plus': {'pxtract_n_isotope': 2}}
[pParse2Plus] 0, C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\L1_20211004_MxR_secondbatch_DSSOplate_B12.raw
[cur] 2026-06-01 23:38:35 Starting...
[pParse2Plus] 0, Extract using pParse
[pParse2Plus] "C:\pFindStudio\pLink\3.0.17\bin\pParse.exe" -D "C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\L1_20211004_MxR_secondbatch_DSSOplate_B12.raw" -t -0.8 -z 5 -i 1 -C 1 -a 0 -p 1 -S 1 -O "C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui"

  *********************************************
 /       pParse2.0 (x64) from pFind Studio     \
/        Email  :  pfind@ict.ac.cn              \
\        Website:  http://pfind.ict.ac.cn       /
  *********************************************

[pParse] Calling pXtract to export MS1 and MS2 files...
[pXtract] The software will expire in 01/01/2100
[pXtract] Begin to extract C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\L1_20211004_MxR_secondbatch_DSSOplate_B12.raw
[pXtract] pXtract tries to use MSFileReader
[pXtract] : 100.00%
[pXtract] Output Files Completed!
[pXtract] == == == Time elapsed: 13.692s. == == ==
[pParse] MS1list completes.
[pParse] Processing L1_20211004_MxR_secondbatch_DSSOplate_B12.MS1
[pParse] Loading MS1 and MS2 files...
[pParse] Loading MS1 Spectra Number: 18633
[pParse] Loading MS2 Spectra Number: 28141
[pParse] Writing MS1 to PF...

[pParse] : 100%

[pParse] Evaluting by the classifier...
[pParse] Filtering by the threshold...

[pParse] : 100%
[pParse] Writing CSV...
[pParse] Releasing resources...
[pParse] Saving CSV to disk...
[pParse] Exported 128961 precursors.
[pParse] Found 0 duplicate scans.
[pParse] == == == Time elapsed: 25 seconds. == == == =
[pParse2Plus] 0, Load pXtract ms2 head
[pParse2Plus] 0, Load pParse2 mgf head
[pParse2Plus] 0, Merge precursor
[pParse2Plus] 0, # merge precursor: 137424, # scan: 28141, multiplier: 4.88
[pParse2Plus] 0, # precursor from distribution: {1: 107599, 2: 21362, 0: 8463}
[pParse2Plus] 0, Calculate precursor score
[pParse2Plus] 0, # precursor score distribution: {0: 0.2558, 1: 0.3646, 2: 0.3423}
[pParse2Plus] 0, Write mgf
[ERROR] Traceback (most recent call last):
File "pParse2Plus.py", line 96, in
File "pParse2Plus.py", line 79, in _main
File "flow.py", line 386, in flow.arg_plink_cfg
File "flow.py", line 339, in flow.flow_pparse2plus_cfg
File "flow.py", line 300, in flow.flow_raw_cfg
File "mgf_file.py", line 45, in mgf_file.write_mgf_from_ms2_mp
File "mgf_file.py", line 55, in mgf_file.write_mgf_from_ms2_mp
KeyError: 3324
[12872] Failed to execute script 'pParse2Plus' due to unhandled exception!

[pLink] Welcome to use pLink v3.0.17, it will be expired on 20280110
[pLink] Search Engine initializing...
[pLink] Generating reverse database...
[pLink] Search identifier: GPVSP
[pLink] Search Engine is ready to search.

[pLink] Start searching C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\L1_20211004_MxR_secondbatch_DSSOplate_B12_HCDFT.pf2, Labeling None
[pLink] Total spectra: 128961
[pLink] Loaded 5000 spectra, 5000 / 128961
[pLink] Loaded 5000 spectra, 10000 / 128961
[pLink] Loaded 5000 spectra, 15000 / 128961
[pLink] Loaded 5000 spectra, 20000 / 128961
[pLink] Loaded 5000 spectra, 25000 / 128961
[pLink] Loaded 5000 spectra, 30000 / 128961
[pLink] Loaded 5000 spectra, 35000 / 128961
[pLink] Loaded 5000 spectra, 40000 / 128961
[pLink] Loaded 5000 spectra, 45000 / 128961
[pLink] Loaded 5000 spectra, 50000 / 128961
[pLink] Loaded 5000 spectra, 55000 / 128961
[pLink] Loaded 5000 spectra, 60000 / 128961
[pLink] Loaded 5000 spectra, 65000 / 128961
[pLink] Loaded 5000 spectra, 70000 / 128961
[pLink] Loaded 5000 spectra, 75000 / 128961
[pLink] Loaded 5000 spectra, 80000 / 128961
[pLink] Loaded 5000 spectra, 85000 / 128961
[pLink] Loaded 5000 spectra, 90000 / 128961
[pLink] Loaded 5000 spectra, 95000 / 128961
[pLink] Loaded 5000 spectra, 100000 / 128961
[pLink] Loaded 5000 spectra, 105000 / 128961
[pLink] Loaded 5000 spectra, 110000 / 128961
[pLink] Loaded 5000 spectra, 115000 / 128961
[pLink] Loaded 5000 spectra, 120000 / 128961
[pLink] Loaded 5000 spectra, 125000 / 128961
[pLink] Loaded 3961 spectra, 128961 / 128961
[pLink] Complete First search.

[pLink] Load File0.Tmp.All.pfd.
[pLink] Start re-ranking...
[pLink] Iteration: round 1/5
[pLink] Iteration: round 2/5
[pLink] Iteration: round 3/5
[pLink] Iteration: round 4/5
[pLink] Iteration: round 5/5
[RerankOut] C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\failed_file_example_gui\failed_file_example_gui.plink
[RerankOut] Start...
[RerankOut] Model hyperparameter optimization...
[RerankOut] Iteration: round 1/5
[RerankOut] Iteration: round 2/5
[RerankOut] Iteration: round 3/5
[RerankOut] Iteration: round 4/5
[RerankOut] Iteration: round 5/5
[pLink] Complete re-rank.

[pLink] Saved result_2026.06.01.File0.pfd.
[pLink] Complete Searching L1_20211004_MxR_secondbatch_DSSOplate_B12_HCDFT.pf2.

[pLink] Start to generate reports...
[pLink] Controlling FDR at the peptide level...
[pLink] Complete report.
[pLink] Start to generate filter reports...
[pLinkFDR] C:\Users\BittremieuxLab\Documents\CasanovoXL\failed_file_example_gui\failed_file_example_gui\failed_file_example_gui.plink
[pLinkFDR] Start...
[pLinkFDR] Filtering...
[pLink] Load the titltes to filter.
[pLink] Complete report.

pParsePlusLog.txt

failed_file_example_gui.log
pLinkFDR.log
RerankOut.log

failed_file_example_gui_plink.txt

log_out.txt

[NorthblueM]

Hi MichelDierckx,

You may have selected the wrong MS Data Format.

The first option, RAW (Orbitrap), uses Thermo’s official MSFileReader to read RAW files. However, official maintenance and updates for MSFileReader were discontinued since 2017. We kept this option only so that some users to reproduce their previous data analyses. In addition, the data preprocessing under this option does not natively support RAW files with FAIMS, while the data you are running includes FAIMS.

The second option, RAW (FAIMS)(Orbitrap New Instrument, Ascend), uses Thermo’s official RawFileReader to read RAW files. Under this option, pParse2Plus natively supports RAW files with FAIMS. We tested the data you mentioned, PXD042173/L1_20211004_MxR_secondbatch_DSSOplate_B12.raw, and the search ran without any issues.

Therefore, we suggest selecting the second MS Data Format option in the GUI. For more details, you can refer to Choosing MS Data Format.

In addition, please note that when you rerun the analysis, you should place the RAW files in a new and clean folder. In other words, please delete all intermediate files except the RAW files to avoid conflicts between the new run and the previously extracted spectra files.

Best,
Pengzhi