Lots of G's in X!Tandem screen output

[StevenVerbruggen]

Hi Marc and Harald,

I am currently reanalyzing melanoma data (the test I describe here, is performed on file 20141212_QEp7_MiBa_SA_HLA-I-p_MM16_1_A.raw of ProteomeXChange dataset PXD004894). The RAW file was first converted to mgf and mzML using ThermoRawFileParser (http://compomics.github.io/projects/ThermoRawFileParser).

I use the mgf file in an X!Tandem search:

nohup java -cp /home/steven/tools/SearchGUI-4.0.18/SearchGUI-4.0.18.jar eu.isas.searchgui.cmd.SearchCLI -spectrum_files /data2/steven/melanoma_proteomics_bassinisternberg/mel16/mgf/20141212_QEp7_MiBa_SA_HLA-I-p_MM16_1_A.mgf -fasta_file /data2/steven/melanoma_proteomics_bassinisternberg/mel16/fasta/comb_fasta_rnaseq_uniprot_hspvdb_filtered_crap_concatenated_target_decoy.fasta -output_folder /data2/steven/melanoma_proteomics_bassinisternberg/mel16/xtandem_out/ -id_params /data2/steven/melanoma_proteomics_bassinisternberg/mel16/bassinisternberg_mel16.par -xtandem 1 -comet 1 -threads 5 > nohup_searchgui_xtandem_comet.txt &

This generates following screen output in the nohup file:

Thu Apr 22 15:26:15 CEST 2021 Importing spectrum files.
Thu Apr 22 15:26:15 CEST 2021 Importing spectrum files completed (108.0 milliseconds).

Processing: 20141212_QEp7_MiBa_SA_HLA-I-p_MM16_1_A.mgf (1/1) 
Thu Apr 22 15:26:15 CEST 2021 Converting spectrum file 20141212_QEp7_MiBa_SA_HLA-I-p_MM16_1_A.mgf to peak list. 
10% 20% 30% 40% 50% 60% 70% 80% 90%

xtandem command: 
/home/steven/tools/SearchGUI-4.0.18/resources/XTandem/linux/linux_64bit/tandem /home/steven/tools/SearchGUI-4.0.18/resources/temp/search_engines/xtandem/input_searchGUI.xml 

Thu Apr 22 15:26:30 CEST 2021 Processing 20141212_QEp7_MiBa_SA_HLA-I-p_MM16_1_A.mgf with X!Tandem.

X! TANDEM Vengeance (2015.12.15.2)

Loading spectra (mgf).............................. loaded.
Spectra matching criteria = 58797
Starting threads ..... started. 
Computing models:
G
G
G
G
G
G
G
G
G
G
G
G

The list of new lines with a 'G' keeps running for quite a while and grows while the search is running. The normal progress messages are not to be found in the screen output. Any idea what can cause this stream of G's? I used X!Tandem in SearchGUI 4.0.18 before on other data and that gave the normal screen output. I search with X!Tandem on the mzML file as well, gave the same phenomenon. Searching the mzML file with MSGF+ in SearchGUI seems to react normal (although the run is currently still running, but I get the expected progress messages over there: work splitted in 15 tasks, 1.78% currently complete). I also included the used parameter file here (extension was changed from 'par' to 'txt' for GitHub upload guidelines). bassinisternberg_mel16.txt

I performed the X!Tandem search last week on an mgf conversion of the same RAW file, but that time generated with MSConvert. Gave also the stream of G's and I let that search running for 5 days without any progress messages in the stream of G's (did a quick search through the nohup, only G's) nor any end result. This project is done for a peptidomics project and we saw better results with X!Tandem than with MSGF+ earlier, so would be nice to r

Thanks for any insight into this problem in advance. If you would need any more info, please let me know.

Best regards, Steven

[hbarsnes]

Hi Steven,

The long list of G's in the X!Tandem output usually indicates an issue with the search settings. The problem here seems to be the use of the "Acetylation of peptide N-term". I will dig a bit deeper and get back to you. But for now you should be able to simply remove the "Acetylation of peptide N-term" modification when running X!Tandem, as X!Tandem by default searches for these in its second pass search anyway.

We do not have a lot of experience with non-enzymatic searches though, so there may be other X!Tandem parameters that you want to fine tune as well.

Best regards, Harald

[hbarsnes]

Hi again,

I have to take back that point about X!Tandem adding the "Acetylation of peptide N-term" in its second pass search. That is only for "protein N-term" (https://www.thegpm.org/TANDEM/api/pqa.html).

But in any case, it would be great if you could try without "Acetylation of peptide N-term" just to see if the search then completes? At least there are no G's printed on my end after this change.

Best regards, Harald

[StevenVerbruggen]

Hi Harald,

Thanks for your swift input. It was actually the purpose to include "Acetylation of protein N-term" as a variable mod instead of "Acetylation of peptide N-term". Appears that I copied the wrong line from the mods list. I started an X!Tandem run now with protein N-term acetylation in the parameter file instead of peptide N-term acetylation. Half hour in the run and no stream of G's yet. I will keep you updated when the run is completed, but looks good. If it won't work this way after all, I will try another run with neither protein nor peptide N-term acetylation and check what that brings.

Thanks again. Have a nice weekend,

Steven

[hbarsnes]

Hi Steven,

Thanks for the update. In any case, I'm now in the process of releasing a new version that should also allow you to search with the acetylation on the peptide n-term. There was a bug in which we ended up converting the acetylation on the peptide n-term into acetylation on any amino acid, thus explaining why X!Tandem was not very happy. :)

I will close this issue, but don't hesitate to open a new one if you come across other problems.

Best regards, Harald