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Nesvilab / FragPipe

A cross-platform Graphical User Interface (GUI) for running MSFragger and Philosopher - powered pipeline for comprehensive analysis of shotgun proteomics data
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Philosopher 4.4.0 bug: TMT column names don't match those from the annotation files 2 #792

Closed fstein closed 2 years ago

fstein commented 2 years ago

Hello,

when running FragPipe in headless mode (analyzing a TMT experiment), I get weird TMT channel names. I put an example here: https://oc.embl.de/index.php/s/9iEiAtMw6j0hSMK

In the annotation file, channels are named as channel_126, channel_127N and the annotation.txt file looks like the following: 126 channel_126 127N channel_127N 127C channel_127C ...

Now the names in the ion.tsv, peptide.tsv and protein.tsv output are named like channel_126126, channel_126127N, channel_126127C etc. Names in the psm.tsv are as expected (channel_126, channel_127N etc.). It somehow always takes the name of the 126 annotation as named in the annotation.txt and puts it as a prefix in front of the TMT channel name (such as 126, 127N, 127C etc.).

Do you have any idea what I am doing wrong or what the problem might be?

Previously I observed a similar issue which I could resolve when switching from Percolator to PeptideProphet (here only the channel names of the protein.tsv output were named incorrectly as described above).

I put it now in philosopher issues, as Fengchao and Felipe told me that it is related to philosopher.

Thanks a lot for your help.

Best,

Frank

fstein commented 2 years ago

PS: Here is the log file

10:21:09 WARN : The output directory is not empty. Some files might be overwritten in: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4

System OS: Windows 7, Architecture: AMD64 Java Info: 18.0.1.1, Java HotSpot(TM) 64-Bit Server VM, Oracle Corporation

Version info: FragPipe version 18.1-build2 MSFragger version 3.5 Philosopher version 4.4.0

LCMS files: Experiment/Group:

16 commands to execute: CheckCentroid java -Xmx24G -cp "X:\06_scripts\MSFragger_cmd\fragpipe\lib\fragpipe-18.1-build2.jar;X:\06_scripts\MSFragger_cmd\fragpipe\tools\batmass-io-1.25.5.jar" com.dmtavt.fragpipe.util.CheckCentroid X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML 39 WorkspaceCleanInit [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\PROTEO~1\AppData\Local\Temp\e0e0ec55-e2a2-44ce-b1d6-ad8ab326d429 MSFragger [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] java -jar -Dfile.encoding=UTF-8 -Xmx24G X:\06_scripts\MSFragger_cmd\fragpipe\tools\MSFragger-3.5\MSFragger-3.5.jar X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragger.params X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML PeptideProphet: Workspace init [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\PROTEO~1\AppData\Local\Temp\521385ea-be53-4917-9f42-351b95524bdb PeptideProphet [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windowsamd64\philosopher.exe peptideprophet --decoyprobs --ppm --accmass --nonparam --expectscore --decoy rev --database X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas ..\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML PeptideProphet: Delete temp java -cp X:\06_scripts\MSFragger_cmd\fragpipe\lib\fragpipe-18.1-build2.jar com.github.chhh.utils.FileDelete X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp Rewrite pepxml [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] java -cp X:\06_scripts\MSFragger_cmd\fragpipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML ProteinProphet [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\filelist_proteinprophet.txt PhilosopherDbAnnotate [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe database --annotate X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries05082022.fasta.fas --prefix rev PhilosopherFilter [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windowsamd64\philosopher.exe filter --sequential --picked --prot 0.01 --tag rev --pepxml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4 --protxml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\combined.prot.xml --razor FreeQuant [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe freequant --ptw 0.4 --tol 10 --dir X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4 Quant (Isobaric) [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe labelquant --tol 20 --level 2 --plex 6 --annot X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\annotation.txt --brand tmt --dir X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4 PhilosopherReport [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe report WorkspaceClean [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --clean --nocheck TmtIntegrator [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] java -Xmx24G -cp "X:\06_scripts\MSFragger_cmd\fragpipe\tools\tmt-integrator-3.4.jar" TMTIntegrator X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\tmt-integrator-conf.yml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\psm.tsv


Execution order:

    Cmd: [START], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [CheckCentroid], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [WorkspaceCleanInit], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [MSFragger], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [PeptideProphet], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [ProteinProphet], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [PhilosopherDbAnnotate], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [PhilosopherFilter], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [FreeQuant], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [Quant (Isobaric)], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [PhilosopherReport], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [WorkspaceClean], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]
    Cmd: [TmtIntegrator], Work dir: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4]

>contam_sp|O77727|K1C15_SHEEP Keratin, type I cytoskeletal 15 OS=Ovis aries GN=KRT15 PE=2 SV=1
>rev_sp|O75159|SOCS5_HUMAN Suppressor of cytokine signaling 5 OS=Homo sapiens OX=9606 GN=SOCS5 PE=1 SV=1
>rev_sp|P10646|TFPI1_HUMAN Tissue factor pathway inhibitor OS=Homo sapiens OX=9606 GN=TFPI PE=1 SV=1
>rev_sp|P40937-2|RFC5_HUMAN Isoform 2 of Replication factor C subunit 5 OS=Homo sapiens OX=9606 GN=RFC5
>rev_sp|Q11203-26|SIAT6_HUMAN Isoform E3+32 of CMP-N-acetylneuraminate-beta-1,4-galactoside alpha-2,3-sialyltransferase OS=Homo sapiens OX=9606 GN=ST3GAL3
>rev_sp|Q5SRH9-6|TT39A_HUMAN Isoform 6 of Tetratricopeptide repeat protein 39A OS=Homo sapiens OX=9606 GN=TTC39A
>rev_sp|Q7Z614-4|SNX20_HUMAN Isoform 4 of Sorting nexin-20 OS=Homo sapiens OX=9606 GN=SNX20
>rev_sp|Q8TAA1|RNS11_HUMAN Probable ribonuclease 11 OS=Homo sapiens OX=9606 GN=RNASE11 PE=1 SV=1
>rev_sp|Q99440|CD006_HUMAN Uncharacterized protein encoded by LINC01587 OS=Homo sapiens OX=9606 GN=LINC01587 PE=2 SV=1
>rev_sp|Q9NSV4-6|DIAP3_HUMAN Isoform 6 of Protein diaphanous homolog 3 OS=Homo sapiens OX=9606 GN=DIAPH3
>rev_sp|Q9Y6X4|F169A_HUMAN Soluble lamin-associated protein of 75 kDa OS=Homo sapiens OX=9606 GN=FAM169A PE=1 SV=2
>sp|O75190-4|DNJB6_HUMAN Isoform D of DnaJ homolog subfamily B member 6 OS=Homo sapiens OX=9606 GN=DNAJB6
>sp|P10911-5|MCF2_HUMAN Isoform 5 of Proto-oncogene DBL OS=Homo sapiens OX=9606 GN=MCF2
>sp|P41219|PERI_HUMAN Peripherin OS=Homo sapiens OX=9606 GN=PRPH PE=1 SV=2
>sp|Q12778|FOXO1_HUMAN Forkhead box protein O1 OS=Homo sapiens OX=9606 GN=FOXO1 PE=1 SV=2
>sp|Q5SW79-3|CE170_HUMAN Isoform 3 of Centrosomal protein of 170 kDa OS=Homo sapiens OX=9606 GN=CEP170
>sp|Q7Z6I6-4|RHG30_HUMAN Isoform 4 of Rho GTPase-activating protein 30 OS=Homo sapiens OX=9606 GN=ARHGAP30
>sp|Q8TAF8|LHPL5_HUMAN LHFPL tetraspan subfamily member 5 protein OS=Homo sapiens OX=9606 GN=LHFPL5 PE=1 SV=1
>sp|Q99502-3|EYA1_HUMAN Isoform EYA1D of Eyes absent homolog 1 OS=Homo sapiens OX=9606 GN=EYA1
>sp|Q9NTK1|DEPP1_HUMAN Protein DEPP1 OS=Homo sapiens OX=9606 GN=DEPP1 PE=1 SV=2
>sp|Q9YWQ2|VP10_BAVJK Structural protein VP10 OS=Banna virus (strain Indonesia/JKT-6423/1980) OX=649604 GN=Segment-10 PE=4 SV=1
X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\annotation.txt:
126 channel_126
127N channel_127N
128C channel_128C
129N channel_129N
130C channel_130C
131N channel_131N
# FragPipe v18.1-build2ui state cache

# Please edit the following path to point to the correct location.
# In Windows, please replace single '\' with '\\'
database.db-path=X\:\\01_users\\Raphael\\Vorversuche\\Experiments\\Lysate_test_TMT4\\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas

crystalc.run-crystalc=false
database.decoy-tag=rev_
diann.fragpipe.cmd-opts=
diann.library=
diann.q-value=0.01
diann.quantification-strategy=3
diann.run-dia-nn=false
diann.run-specific-protein-q-value=0.01
diann.unrelated-runs=false
diann.use-predicted-spectra=false
diaumpire.AdjustFragIntensity=true
diaumpire.BoostComplementaryIon=false
diaumpire.CorrThreshold=0
diaumpire.DeltaApex=0.2
diaumpire.ExportPrecursorPeak=false
diaumpire.Q1=true
diaumpire.Q2=true
diaumpire.Q3=true
diaumpire.RFmax=500
diaumpire.RPmax=25
diaumpire.RTOverlap=0.3
diaumpire.SE.EstimateBG=false
diaumpire.SE.IsoPattern=0.3
diaumpire.SE.MS1PPM=10
diaumpire.SE.MS2PPM=20
diaumpire.SE.MS2SN=1.1
diaumpire.SE.MassDefectFilter=true
diaumpire.SE.MassDefectOffset=0.1
diaumpire.SE.NoMissedScan=1
diaumpire.SE.SN=1.1
diaumpire.run-diaumpire=false
fragpipe-config.bin-ionquant=\\\\pcf.embl.de\\pcf\\06_scripts\\MSFragger_cmd\\fragpipe\\tools\\IonQuant-1.8.0.jar
fragpipe-config.bin-msfragger=X\:\\06_scripts\\MSFragger_cmd\\fragpipe\\tools\\MSFragger-3.5\\MSFragger-3.5.jar
fragpipe-config.bin-philosopher=X\:\\06_scripts\\MSFragger_cmd\\fragpipe\\tools\\philosopher_v4.4.0_windows_amd64\\philosopher.exe
fragpipe-config.bin-python=
freequant.mz-tol=10
freequant.rt-tol=0.4
freequant.run-freequant=false
ionquant.excludemods=
ionquant.heavy=
ionquant.imtol=0.05
ionquant.ionfdr=0.01
ionquant.light=
ionquant.locprob=0.75
ionquant.maxlfq=1
ionquant.mbr=0
ionquant.mbrimtol=0.05
ionquant.mbrmincorr=0
ionquant.mbrrttol=1
ionquant.mbrtoprun=100000
ionquant.medium=
ionquant.minexps=1
ionquant.minfreq=0
ionquant.minions=2
ionquant.minisotopes=2
ionquant.minscans=3
ionquant.mztol=10
ionquant.normalization=1
ionquant.peptidefdr=1
ionquant.proteinfdr=1
ionquant.requantify=1
ionquant.rttol=0.4
ionquant.run-ionquant=true
ionquant.tp=0
ionquant.uniqueness=0
ionquant.use-labeling=false
ionquant.use-lfq=true
ionquant.writeindex=0
msbooster.predict-rt=true
msbooster.predict-spectra=true
msbooster.run-msbooster=false
msbooster.use-correlated-features=false
msfragger.Y_type_masses=
msfragger.activation_types=all
msfragger.add_topN_complementary=0
msfragger.allowed_missed_cleavage_1=2
msfragger.allowed_missed_cleavage_2=2
msfragger.calibrate_mass=2
msfragger.check_spectral_files=true
msfragger.clip_nTerm_M=true
msfragger.deisotope=1
msfragger.delta_mass_exclude_ranges=(-1.5,3.5)
msfragger.deneutralloss=1
msfragger.diagnostic_fragments=
msfragger.diagnostic_intensity_filter=0
msfragger.digest_max_length=50
msfragger.digest_min_length=7
msfragger.fragment_ion_series=b,y
msfragger.fragment_mass_tolerance=20
msfragger.fragment_mass_units=1
msfragger.intensity_transform=0
msfragger.ion_series_definitions=
msfragger.isotope_error=-1/0/1/2/3
msfragger.labile_search_mode=off
msfragger.localize_delta_mass=false
msfragger.mass_diff_to_variable_mod=0
msfragger.mass_offsets=0
msfragger.max_fragment_charge=2
msfragger.max_variable_mods_combinations=5000
msfragger.max_variable_mods_per_peptide=3
msfragger.min_fragments_modelling=2
msfragger.min_matched_fragments=4
msfragger.min_sequence_matches=2
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.01
msfragger.misc.fragger.clear-mz-hi=131.5
msfragger.misc.fragger.clear-mz-lo=125.5
msfragger.misc.fragger.digest-mass-hi=5000
msfragger.misc.fragger.digest-mass-lo=200
msfragger.misc.fragger.enzyme-dropdown-1=trypsin
msfragger.misc.fragger.enzyme-dropdown-2=null
msfragger.misc.fragger.precursor-charge-hi=6
msfragger.misc.fragger.precursor-charge-lo=1
msfragger.misc.fragger.remove-precursor-range-hi=1.5
msfragger.misc.fragger.remove-precursor-range-lo=-1.5
msfragger.misc.slice-db=1
msfragger.num_enzyme_termini=2
msfragger.output_format=pepXML_pin
msfragger.output_max_expect=50
msfragger.output_report_topN=1
msfragger.override_charge=false
msfragger.precursor_mass_lower=-20
msfragger.precursor_mass_mode=selected
msfragger.precursor_mass_units=1
msfragger.precursor_mass_upper=20
msfragger.precursor_true_tolerance=20
msfragger.precursor_true_units=1
msfragger.remainder_fragment_masses=
msfragger.remove_precursor_peak=1
msfragger.report_alternative_proteins=true
msfragger.restrict_deltamass_to=all
msfragger.run-msfragger=true
msfragger.search_enzyme_cut_1=KR
msfragger.search_enzyme_cut_2=
msfragger.search_enzyme_name_1=trypsin
msfragger.search_enzyme_name_2=null
msfragger.search_enzyme_nocut_1=P
msfragger.search_enzyme_nocut_2=
msfragger.search_enzyme_sense_1=C
msfragger.search_enzyme_sense_2=C
msfragger.table.fix-mods=0.000000,C-Term Peptide,true,-1; 0.000000,N-Term Peptide,true,-1; 0.000000,C-Term Protein,true,-1; 0.000000,N-Term Protein,true,-1; 0.000000,G (glycine),true,-1; 0.000000,A (alanine),true,-1; 0.000000,S (serine),true,-1; 0.000000,P (proline),true,-1; 0.000000,V (valine),true,-1; 0.000000,T (threonine),true,-1; 57.021460,C (cysteine),true,-1; 0.000000,L (leucine),true,-1; 0.000000,I (isoleucine),true,-1; 0.000000,N (asparagine),true,-1; 0.000000,D (aspartic acid),true,-1; 0.000000,Q (glutamine),true,-1; 229.162932,K (lysine),true,-1; 0.000000,E (glutamic acid),true,-1; 0.000000,M (methionine),true,-1; 0.000000,H (histidine),true,-1; 0.000000,F (phenylalanine),true,-1; 0.000000,R (arginine),true,-1; 0.000000,Y (tyrosine),true,-1; 0.000000,W (tryptophan),true,-1; 0.000000,B ,true,-1; 0.000000,J,true,-1; 0.000000,O,true,-1; 0.000000,U,true,-1; 0.000000,X,true,-1; 0.000000,Z,true,-1
msfragger.table.var-mods=15.994900,M,true,3; 42.010600,[^,true,1; 229.162932,n^,true,1
msfragger.track_zero_topN=0
msfragger.use_all_mods_in_first_search=false
msfragger.use_topN_peaks=300
msfragger.write_calibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=true
percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc
percolator.keep-tsv-files=false
percolator.min-prob=0.5
percolator.run-percolator=false
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--sequential --picked --prot 0.01
phi-report.pep-level-summary=false
phi-report.philosoher-msstats=false
phi-report.print-decoys=false
phi-report.prot-level-summary=false
phi-report.run-report=true
protein-prophet.cmd-opts=--maxppmdiff 2000000 --minprob 0.5
protein-prophet.run-protein-prophet=true
ptmprophet.cmdline=
ptmprophet.run-ptmprophet=false
ptmshepherd.adv_params=false
ptmshepherd.annotation-common=false
ptmshepherd.annotation-custom=false
ptmshepherd.annotation-glyco=false
ptmshepherd.annotation-unimod=true
ptmshepherd.annotation_file=
ptmshepherd.annotation_tol=0.01
ptmshepherd.assign_glycans=true
ptmshepherd.cap_y_ions=
ptmshepherd.decoy_type=1
ptmshepherd.diag_ions=
ptmshepherd.diagextract_mode=false
ptmshepherd.diagmine_diagMinFoldChange=3.0
ptmshepherd.diagmine_diagMinSpecDiff=00.2
ptmshepherd.diagmine_fragMinFoldChange=3.0
ptmshepherd.diagmine_fragMinPropensity=00.1
ptmshepherd.diagmine_fragMinSpecDiff=00.1
ptmshepherd.diagmine_minIons=25
ptmshepherd.diagmine_minIonsPerSpec=2
ptmshepherd.diagmine_minPeps=25
ptmshepherd.diagmine_mode=false
ptmshepherd.diagmine_pepMinFoldChange=3.0
ptmshepherd.diagmine_pepMinSpecDiff=00.2
ptmshepherd.glyco_adducts=
ptmshepherd.glyco_fdr=1.00
ptmshepherd.glyco_isotope_max=3
ptmshepherd.glyco_isotope_min=-1
ptmshepherd.glyco_mode=false
ptmshepherd.glyco_ppm_tol=50
ptmshepherd.glycodatabase=
ptmshepherd.histo_smoothbins=2
ptmshepherd.iontype_a=false
ptmshepherd.iontype_b=true
ptmshepherd.iontype_c=true
ptmshepherd.iontype_x=false
ptmshepherd.iontype_y=true
ptmshepherd.iontype_z=true
ptmshepherd.localization_allowed_res=
ptmshepherd.localization_background=4
ptmshepherd.max_adducts=0
ptmshepherd.n_glyco=true
ptmshepherd.normalization-psms=true
ptmshepherd.normalization-scans=false
ptmshepherd.output_extended=false
ptmshepherd.peakpicking_mass_units=0
ptmshepherd.peakpicking_minPsm=10
ptmshepherd.peakpicking_promRatio=0.3
ptmshepherd.peakpicking_width=0.002
ptmshepherd.precursor_mass_units=0
ptmshepherd.precursor_tol=0.01
ptmshepherd.print_decoys=false
ptmshepherd.prob_dhexOx=
ptmshepherd.prob_dhexY=
ptmshepherd.prob_neuacOx=
ptmshepherd.prob_neugcOx=
ptmshepherd.prob_phosphoOx=
ptmshepherd.prob_regY=
ptmshepherd.prob_sulfoOx=
ptmshepherd.remainder_masses=
ptmshepherd.remove_glycan_delta_mass=true
ptmshepherd.run-shepherd=false
ptmshepherd.spectra_maxfragcharge=2
ptmshepherd.spectra_ppmtol=20
ptmshepherd.varmod_masses=
quantitation.run-label-free-quant=false
run-psm-validation=true
run-validation-tab=true
speclibgen.easypqp.extras.max_delta_ppm=15
speclibgen.easypqp.extras.max_delta_unimod=0.02
speclibgen.easypqp.extras.rt_lowess_fraction=0
speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM
speclibgen.easypqp.rt-cal=ciRT
speclibgen.easypqp.select-file.text=
speclibgen.easypqp.select-im-file.text=
speclibgen.keep-intermediate-files=false
speclibgen.run-speclibgen=false
tmtintegrator.add_Ref=1
tmtintegrator.aggregation_method=1
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.glyco_qval=-1
tmtintegrator.groupby=0
tmtintegrator.max_pep_prob_thres=0.9
tmtintegrator.min_ntt=0
tmtintegrator.min_pep_prob=0.9
tmtintegrator.min_percent=0.05
tmtintegrator.min_purity=0.5
tmtintegrator.min_site_prob=-1
tmtintegrator.mod_tag=none
tmtintegrator.ms1_int=true
tmtintegrator.outlier_removal=true
tmtintegrator.print_RefInt=false
tmtintegrator.prot_exclude=none
tmtintegrator.prot_norm=0
tmtintegrator.psm_norm=false
tmtintegrator.quant_level=2
tmtintegrator.ref_tag=channel_126
tmtintegrator.run-tmtintegrator=true
tmtintegrator.tolerance=20
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=1
tmtintegrator.unique_pep=true
tmtintegrator.use_glycan_composition=false
workdir=X\:\\01_users\\Raphael\\Vorversuche\\Experiments\\Lysate_test_TMT4
workflow.input.data-type.im-ms=false
workflow.input.data-type.regular-ms=true
workflow.ram=0
workflow.threads=39

CheckCentroid java -Xmx24G -cp "X:\06_scripts\MSFragger_cmd\fragpipe\lib\fragpipe-18.1-build2.jar;X:\06_scripts\MSFragger_cmd\fragpipe\tools\batmass-io-1.25.5.jar" com.dmtavt.fragpipe.util.CheckCentroid X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML 39 Process 'CheckCentroid' finished, exit code: 0 Done in 6432 ms. WorkspaceCleanInit [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --clean --nocheck INFO[10:21:16] Executing Workspace v4.4.0
INFO[10:21:16] Removing workspace
INFO[10:21:16] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\PROTEO~1\AppData\Local\Temp\e0e0ec55-e2a2-44ce-b1d6-ad8ab326d429 INFO[10:21:17] Executing Workspace v4.4.0
INFO[10:21:17] Creating workspace
INFO[10:21:17] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] java -jar -Dfile.encoding=UTF-8 -Xmx24G X:\06_scripts\MSFragger_cmd\fragpipe\tools\MSFragger-3.5\MSFragger-3.5.jar X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragger.params X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML MSFragger version MSFragger-3.5 Batmass-IO version 1.25.5 timsdata library version timsdata-2-8-7-1 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. System OS: Windows 7, Architecture: AMD64 Java Info: 18.0.1.1, Java HotSpot(TM) 64-Bit Server VM, Oracle Corporation JVM started with 24 GB memory Checking database... Checking spectral files... X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML: Scans = 46464 ***FIRST SEARCH**** Parameters: num_threads = 39 database_name = X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 20.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mgf = 0 isotope_error = 0/1 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = trypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = P allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = pepxml_pin output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = false override_charge = false precursor_charge_low = 1 precursor_charge_high = 6 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 200.0 digest_mass_range_high = 5000.0 max_fragment_charge = 1 deisotope = 1 deneutralloss = true track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 300 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.01 intensity_transform = 0 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 125.5 clear_mz_range_high = 131.5 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.994900 M 3 variable_mod_02 = 42.010600 [^ 1 variable_mod_03 = 229.162932 n^ 1 add_A_alanine = 0.000000 add_B_user_amino_acid = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_J_user_amino_acid = 0.000000 add_K_lysine = 229.162932 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.000000 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_U_user_amino_acid = 0.000000 # U = selenocysteine (150.95363 Da) add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_X_user_amino_acid = 0.000000 add_Y_tyrosine = 0.000000 add_Z_user_amino_acid = 0.000000 Number of unique peptides of length 7: 303876 of length 8: 296726 of length 9: 292420 of length 10: 281062 of length 11: 269493 of length 12: 258141 of length 13: 253380 of length 14: 237594 of length 15: 228730 of length 16: 214992 of length 17: 203000 of length 18: 196793 of length 19: 191186 of length 20: 175550 of length 21: 168567 of length 22: 159513 of length 23: 149507 of length 24: 143684 of length 25: 135137 of length 26: 127245 of length 27: 121805 of length 28: 118524 of length 29: 107768 of length 30: 102277 of length 31: 96334 of length 32: 91835 of length 33: 86629 of length 34: 80444 of length 35: 74852 of length 36: 72825 of length 37: 67042 of length 38: 62832 of length 39: 57539 of length 40: 54776 of length 41: 51558 of length 42: 46779 of length 43: 40730 of length 44: 32663 of length 45: 24640 of length 46: 16363 of length 47: 9945 of length 48: 5681 of length 49: 2940 of length 50: 1619 In total 5714996 peptides. Generated 17707829 modified peptides. Number of peptides with more than 5000 modification patterns: 0 Selected fragment index width 0.10 Da. 744971344 fragments to be searched in 1 slices (11.10 GB total) Operating on slice 1 of 1: Fragment index slice generated in 13.36 s

  1. Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML 7.4 s | deisotoping 2.6 s [progress: 46148/46148 (100%) - 8188 spectra/s] 5.6s | postprocessing 0.8 s ***FIRST SEARCH DONE IN 0.881 MIN**
**MASS CALIBRATION AND PARAMETER OPTIMIZATION ----- --------------- --------------- --------------- --------------- MS1 (Old) MS1 (New) MS2 (Old) MS2 (New)
Run Median MAD Median MAD Median MAD Median MAD
001 0.37 1.25 0.03 1.04 -1.31 1.14 -0.09 1.12
----- --------------- --------------- --------------- ---------------
Finding the optimal parameters: ------- ------- ------- ------- ------- ------- ------- ------- ------- MS2 5 7 10 15 20 25 30 50
Count 30534 30649 30739 30711 skip rest
------- ------- ------- ------- ------- ------- ------- ------- -------
------- ------- ------- ------- ------- ------- -------
Peaks 300_0 200_0 175_0 150_1 125_1 100_1
------- ------- ------- ------- ------- ------- -------
Count 30771 30767 skip rest
------- ------- ------- ------- ------- ------- -------
------- -------
Int. 1
------- -------
Count 30897
------- -------
------- -------
Rm P. 0
------- -------
Count 30850
------- -------

New fragment_mass_tolerance = 10 PPM New use_topN_peaks = 300 New minimum_ratio = 0.000000 New intensity_transform = 1 New remove_precursor_peak = 1 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 4.770 MIN*****

****MAIN SEARCH**** Checking database... Parameters: num_threads = 39 database_name = X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 10.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mgf = 0 isotope_error = -1/0/1/2/3 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = trypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = P allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = pepxml_pin output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = true override_charge = false precursor_charge_low = 1 precursor_charge_high = 6 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 200.0 digest_mass_range_high = 5000.0 max_fragment_charge = 1 deisotope = 1 deneutralloss = true track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 300 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.0 intensity_transform = 1 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 125.5 clear_mz_range_high = 131.5 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.994900 M 3 variable_mod_02 = 42.010600 [^ 1 variable_mod_03 = 229.162932 n^ 1 add_A_alanine = 0.000000 add_B_user_amino_acid = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_J_user_amino_acid = 0.000000 add_K_lysine = 229.162932 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.000000 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_U_user_amino_acid = 0.000000 # U = selenocysteine (150.95363 Da) add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_X_user_amino_acid = 0.000000 add_Y_tyrosine = 0.000000 add_Z_user_amino_acid = 0.000000 Number of unique peptides of length 7: 303876 of length 8: 296726 of length 9: 292420 of length 10: 281062 of length 11: 269493 of length 12: 258141 of length 13: 253380 of length 14: 237594 of length 15: 228730 of length 16: 214992 of length 17: 203000 of length 18: 196793 of length 19: 191186 of length 20: 175550 of length 21: 168567 of length 22: 159513 of length 23: 149507 of length 24: 143684 of length 25: 135137 of length 26: 127245 of length 27: 121805 of length 28: 118524 of length 29: 107768 of length 30: 102277 of length 31: 96334 of length 32: 91835 of length 33: 86629 of length 34: 80444 of length 35: 74852 of length 36: 72825 of length 37: 67042 of length 38: 62832 of length 39: 57539 of length 40: 54776 of length 41: 51558 of length 42: 46779 of length 43: 40730 of length 44: 32663 of length 45: 24640 of length 46: 16363 of length 47: 9945 of length 48: 5681 of length 49: 2940 of length 50: 1619 In total 5714996 peptides. Generated 17707829 modified peptides. Number of peptides with more than 5000 modification patterns: 0 Selected fragment index width 0.05 Da. 744971344 fragments to be searched in 1 slices (11.10 GB total) Operating on slice 1 of 1: Fragment index slice generated in 15.11 s

  1. Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzBIN_calibrated 8.1 s [progress: 45984/45984 (100%) - 8394 spectra/s] 5.5s | ramaping alternative proteins and postprocessing 16.3 s MAIN SEARCH DONE IN 0.978 MIN

***TOTAL TIME 6.630 MIN**** Process 'MSFragger' finished, exit code: 0 PeptideProphet: Workspace init [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\PROTEO~1\AppData\Local\Temp\521385ea-be53-4917-9f42-351b95524bdb INFO[10:27:56] Executing Workspace v4.4.0
INFO[10:27:56] Creating workspace
INFO[10:27:56] Done
Process 'PeptideProphet: Workspace init' finished, exit code: 0 PeptideProphet [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windowsamd64\philosopher.exe peptideprophet --decoyprobs --ppm --accmass --nonparam --expectscore --decoy rev --database X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas ..\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML INFO[10:27:57] Executing PeptideProphet v4.4.0
file 1: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML processed altogether 44060 results INFO: Results written to file: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml

using Accurate Mass Bins using PPM mass difference Using Decoy Label "rev_". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling) adding ACCMASS mixture distribution using search_offsets in ACCMASS mixture distr: 0 init with X! Tandem trypsin PeptideProphet (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB read in 0 1+, 26053 2+, 13596 3+, 4411 4+, 0 5+, 0 6+, and 0 7+ spectra. Found 5058 Decoys, and 39002 Non-Decoys MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... Initialising statistical models ... Iterations: .........10.........20.....WARNING: Mixture model quality test failed for charge (1+).

WARNING: Mixture model quality test failed for charge (5+). WARNING: Mixture model quality test failed for charge (6+). WARNING: Mixture model quality test failed for charge (7+). model complete after 26 iterations Process 'PeptideProphet' finished, exit code: 0 PeptideProphet: Delete temp INFO[10:49:15] Done
java -cp X:\06_scripts\MSFragger_cmd\fragpipe\lib\fragpipe-18.1-build2.jar com.github.chhh.utils.FileDelete X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp Process 'PeptideProphet: Delete temp' finished, exit code: 0 Rewrite pepxml [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] java -cp X:\06_scripts\MSFragger_cmd\fragpipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML Fixing pepxml: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml Writing output to: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml15049379343270571543.temp-rewrite Deleting file: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml Moving rewritten file to original location: [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml15049379343270571543.temp-rewrite] -> [X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml] Process 'Rewrite pepxml' finished, exit code: 0 ProteinProphet [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\filelist_proteinprophet.txt INFO[10:49:22] Executing ProteinProphet v4.4.0
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64)) (no FPKM) (no groups) (using degen pep info) Reading in X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml... ...read in 0 1+, 19911 2+, 10341 3+, 2304 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.5

Initializing 28047 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating protein lengths and molecular weights from database x:/01_users/Raphael/Vorversuche/Experiments/Lysate_test_TMT4/2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas .........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........14000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........15000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........16000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........17000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........18000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........19000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........20000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........21000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........22000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........23000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........24000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........25000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........26000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........27000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........28000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........29000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........30000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........31000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........32000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........33000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........34000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........35000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........36000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........37000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........38000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........39000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........40000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........41000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........42000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........43000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........44000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........45000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........46000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........47000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........48000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........49000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........50000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........51000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........52000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........53000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........54000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........55000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........56000 .........:.........:.........:.........:.........:.........:.........:.........:.........:..WARNING: Found the following zero-mass residues in protein entry sp|P07203|GPX1_HUMAN : U MCAARLAAAAAAAQSVYAFSARPLAGGEPVSLGSLRGKVLLIENVASLUGTTVRDYTQMNELQRRLGPRGLVVLGFPCNQFGHQENAKNEEILNSLKYVRPGGGFEPNFMLFEKCEVNGAGAHPLFAFLREALPAPSDDATALMTDPKLITWSPVCRNDVAWNFEKFLVGPDGVPLRRYSRRFQTIDIEPDIEALLSQGPSCA .......57000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........58000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........59000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........60000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........61000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........62000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........63000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........64000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........65000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........66000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........67000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........68000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........69000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........70000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........71000 .........:.........:......WARNING: Found the following zero-mass residues in protein entry sp|Q16881-2|TRXR1_HUMAN : U MLSRLVLNSWAQAIIRPRPPKVLGLQVTTFSEAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-3|TRXR1_HUMAN : U MQQVMLTCKGVNRGHAVPAGPGRKPRPRRSSRLLAGEKHLTRSALLLCHTEDGRALEGTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-4|TRXR1_HUMAN : U MSCEDGRALEGTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-5|TRXR1_HUMAN : U MNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-6|TRXR1_HUMAN : U MPVDDYWLCLPASCARPFVQTVRVVQSCPHCCWFPGVLPSVPEPLRMPAMLPTGSHSAVLPPSHCSTAPPSTSQEPSSSADPKLCLSPPTSDSRQERNVQFGLAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-7|TRXR1_HUMAN : U MVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881|TRXR1_HUMAN : U MGCAEGKAVAAAAPTELQTKGKNGDGRRRSAKDHHPGKTLPENPAGFTSTATADSRALLQAYIDGHSVVIFSRSTCTRCTEVKKLFKSLCVPYFVLELDQTEDGRALEGTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG ...:.........:.........:.........:.........:.........:.........:.........72000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........73000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........74000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........75000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........76000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........77000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........78000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........79000 .........:.........WARNING: Found the following zero-mass residues in protein entry sp|Q86VQ6|TRXR3_HUMAN : U MERSPPQSPGPGKAGDAPNRRSGHVRGARVLSPPGRRARLSSPGPSRSSEAREELRRHLVGLIERSRVVIFSKSYCPHSTRVKELFSSLGVECNVLELDQVDDGARVQEVLSEITNQKTVPNIFVNKVHVGGCDQTFQAYQSGLLQKLLQEDLAYDYDLIIIGGGSGGLSCAKEAAILGKKVMVLDFVVPSPQGTSWGLGGTCVNVGCIPKKLMHQAALLGQALCDSRKFGWEYNQQVRHNWETMTKAIQNHISSLNWGYRLSLREKAVAYVNSYGEFVEHHKIKATNKKGQETYYTAAQFVIATGERPRYLGIQGDKEYCITSDDLFSLPYCPGKTLVVGASYVALECAGFLAGFGLDVTVMVRSILLRGFDQEMAEKVGSYMEQHGVKFLRKFIPVMVQQLEKGSPGKLKVLAKSTEGTETIEGVYNTVLLAIGRDSCTRKIGLEKIGVKINEKSGKIPVNDVEQTNVPYVYAVGDILEDKPELTPVAIQSGKLLAQRLFGASLEKCDYINVPTTVFTPLEYGCCGLSEEKAIEVYKKENLEIYHTLFWPLEWTVAGRENNTCYAKIICNKFDHDRVIGFHILGPNAGEVTQGFAAAMKCGLTKQLLDDTIGIHPTCGEVFTTLEITKSSGLDITQKGCUG :.........:.........:.........:.........:.........:.........:.........:.........80000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........81000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........82000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........83000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........84000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........85000 ........WARNING: Found the following zero-mass residues in protein entry sp|Q92813-2|IOD2_HUMAN : UU MGILSVDLLITLQILPVFFSNCLFLALYDSVILLKHVVLLLSRSKSTRGEWRRMLTSEGLRCVWKSFLLDAYKQLNCPPSGFSKDGHILCLVYEAYKSRLLVYSHLDLWMVKLGEDAPNSSVVHVSSTEGGDNSGNGTQEKIAEGATCHLLDFASPERPLVVNFGSATUPPFTSQLPAFRKLVEEFSSVADFLLVYIDEAHPSDGWAIPGDSSLSFEVKKHQNQEDRCAAAQQLLERFSLPPQCRVVADRMDNNANIAYGVAFERVCIVQRQKIAYLGGKGPFSYNLQEVRHWLEKNFSKRUKKTRLAG .WARNING: Found the following zero-mass residues in protein entry sp|Q92813|IOD2_HUMAN : UU MGILSVDLLITLQILPVFFSNCLFLALYDSVILLKHVVLLLSRSKSTRGEWRRMLTSEGLRCVWKSFLLDAYKQVKLGEDAPNSSVVHVSSTEGGDNSGNGTQEKIAEGATCHLLDFASPERPLVVNFGSATUPPFTSQLPAFRKLVEEFSSVADFLLVYIDEAHPSDGWAIPGDSSLSFEVKKHQNQEDRCAAAQQLLERFSLPPQCRVVADRMDNNANIAYGVAFERVCIVQRQKIAYLGGKGPFSYNLQEVRHWLEKNFSKRUKKTRLAG :.........:.........:.........:.........:.........:.........:.........:.........:.........86000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........87000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........88000 .........:.........:.....WARNING: Found the following zero-mass residues in protein entry sp|Q99611|SPS2_HUMAN : U MAEASATGACGEAMAAAEGSSGPAGLTLGRSFSNYRPFEPQALGLSPSWRLTGFSGMKGUGCKVPQEALLKLLAGLTRPDVRPPLGRGLVGGQEEASQEAGLPAGAGPSPTFPALGIGMDSCVIPLRHGGLSLVQTTDFFYPLVEDPYMMGRIACANVLSDLYAMGITECDNMLMLLSVSQSMSEEEREKVTPLMVKGFRDAAEEGGTAVTGGQTVVNPWIIIGGVATVVCQPNEFIMPDSAVVGDVLVLTKPLGTQVAVNAHQWLDNPERWNKVKMVVSREEVELAYQEAMFNMATLNRTAAGLMHTFNAHAATDITGFGILGHSQNLAKQQRNEVSFVIHNLPIIAKMAAVSKASGRFGLLQGTSAETSGGLLICLPREQAARFCSEIKSSKYGEGHQAWIVGIVEKGNRTARIIDKPRVIEVLPRGATAAVLAPDSSNASSEPSS ....:.........:.........:.........:.........:.........:.........:.........89000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........90000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........91000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........92000 .........:.........:...WARNING: Found the following zero-mass residues in protein entry sp|Q9NNW7-2|TRXR2_HUMAN : U MEDQRGAAAGQRDYDLLVVGGGSGGLACAKEAAQLGRKVAVVDYVEPSPQGTRWGLGGTCVNVGCIPKKLMHQAALLGGLIQDAPNYGWEVAQPVPHDWRKMAEAVQNHVKSLNWGHRVQLQDRKVKYFNIKASFVDEHTVCGVAKGGKEILLSADHIIIATGGRPRYPTHIEGALEYGITSDDIFWLKESPGKTLVVGASYVALECAGFLTGIGLDTTIMMRSIPLRGFDQQMSSMVIEHMASHGTRFLRGCAPSRVRRLPDGQLQVTWEDSTTGKEDTGTFDTVLWAIGRVPDTRSLNLEKAGVDTSPDTQKILVDSREATSVPHIYAIGDVVEGRPELTPIAIMAGRLLVQRLFGGSSDLMDYDNVPTTVFTPLEYGCVGLSEEEAVARHGQEHVEVYHAHYKPLEFTVAGRDASQCYVKMVCLREPPQLVLGLHFLGPNAGEVTQGFALGIKCGASYAQVMRTVGIHPTCSEEVVKLRISKRSGLDPTVTGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q9NNW7|TRXR2_HUMAN : U MAAMAVALRGLGGRFRWRTQAVAGGVRGAARGAAAGQRDYDLLVVGGGSGGLACAKEAAQLGRKVAVVDYVEPSPQGTRWGLGGTCVNVGCIPKKLMHQAALLGGLIQDAPNYGWEVAQPVPHDWRKMAEAVQNHVKSLNWGHRVQLQDRKVKYFNIKASFVDEHTVCGVAKGGKEILLSADHIIIATGGRPRYPTHIEGALEYGITSDDIFWLKESPGKTLVVGASYVALECAGFLTGIGLDTTIMMRSIPLRGFDQQMSSMVIEHMASHGTRFLRGCAPSRVRRLPDGQLQVTWEDSTTGKEDTGTFDTVLWAIGRVPDTRSLNLEKAGVDTSPDTQKILVDSREATSVPHIYAIGDVVEGRPELTPIAIMAGRLLVQRLFGGSSDLMDYDNVPTTVFTPLEYGCVGLSEEEAVARHGQEHVEVYHAHYKPLEFTVAGRDASQCYVKMVCLREPPQLVLGLHFLGPNAGEVTQGFALGIKCGASYAQVMRTVGIHPTCSEEVVKLRISKRSGLDPTVTGCUG ......:.........:.........:.........:.........:.........:.........:.........93000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........94000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........95000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........96000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........97000 .........:.........:.........:.........:.........:.........:.........:.........:.........:......... Total: 97998 Computing degenerate peptides for 12685 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Computing probabilities for 15324 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15324 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15324 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15324 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15324 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15324 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Calculating sensitivity...and error tables... INFO: mu=2.3295e-06, db_size=110651650 Computing MU for 15324 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% INFO[10:49:32] Done
Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe database --annotate X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\2022-08-05-decoys-contam-uniprot_SWP_homo_sapiens_26610entries05082022.fasta.fas --prefix rev INFO[10:49:33] Executing Database v4.4.0
INFO[10:49:33] Annotating the database
INFO[10:49:41] Done
Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windowsamd64\philosopher.exe filter --sequential --picked --prot 0.01 --tag rev --pepxml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4 --protxml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\combined.prot.xml --razor INFO[10:49:41] Executing Filter v4.4.0
INFO[10:49:41] Processing peptide identification files
INFO[10:49:41] Parsing X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml INFO[10:49:48] 1+ Charge profile decoy=0 target=0 INFO[10:49:48] 2+ Charge profile decoy=204 target=20183 INFO[10:49:48] 3+ Charge profile decoy=84 target=10476 INFO[10:49:48] 4+ Charge profile decoy=28 target=2357 INFO[10:49:48] 5+ Charge profile decoy=0 target=0 INFO[10:49:48] 6+ Charge profile decoy=0 target=0 INFO[10:49:48] Database search results ions=28695 peptides=25812 psms=33332 INFO[10:49:48] Converged to 0.95 % FDR with 33016 PSMs decoy=316 threshold=0.0504 total=33332 INFO[10:49:48] Converged to 1.00 % FDR with 25435 Peptides decoy=256 threshold=0.0869 total=25691 INFO[10:49:48] Converged to 1.00 % FDR with 28338 Ions decoy=286 threshold=0.0661 total=28624 INFO[10:49:54] Protein inference results decoy=359 target=6590 INFO[10:49:55] Converged to 1.01 % FDR with 4271 Proteins decoy=43 threshold=0.9256 total=4314 INFO[10:49:57] Applying sequential FDR estimation ions=27878 peptides=25010 psms=32496 INFO[10:49:57] Converged to 0.14 % FDR with 32449 PSMs decoy=47 threshold=0.0525 total=32496 INFO[10:49:57] Converged to 0.16 % FDR with 24969 Peptides decoy=41 threshold=0.5009 total=25010 INFO[10:49:57] Converged to 0.16 % FDR with 27832 Ions decoy=46 threshold=0.0525 total=27878 INFO[10:50:11] Post processing identifications
INFO[10:50:16] Assigning protein identifications to layers
INFO[10:50:17] Processing protein inference
INFO[10:50:46] Synchronizing PSMs and proteins
INFO[10:50:46] Total report numbers after FDR filtering, and post-processing ions=27827 peptides=24964 proteins=4271 psms=32442 INFO[10:50:46] Saving
INFO[10:50:51] Done
Process 'PhilosopherFilter' finished, exit code: 0 FreeQuant [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe freequant --ptw 0.4 --tol 10 --dir X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4 INFO[10:50:51] Executing Label-free quantification v4.4.0
INFO[10:50:54] Indexing PSM information
INFO[10:50:54] Reading spectra and tracing peaks
INFO[10:50:54] Processing Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1 INFO[10:51:26] Assigning intensities to data layers
INFO[10:51:31] Done
Process 'FreeQuant' finished, exit code: 0 Quant (Isobaric) [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe labelquant --tol 20 --level 2 --plex 6 --annot X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\annotation.txt --brand tmt --dir X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4 INFO[10:51:32] Executing Isobaric-label quantification v4.4.0 INFO[10:51:34] Calculating intensities and ion interference INFO[10:51:34] Processing Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1 INFO[10:52:09] Filtering spectra for label quantification
INFO[10:52:09] Removing 0 PSMs from isobaric quantification INFO[10:52:09] Calculating normalized protein levels
INFO[10:52:09] Saving
INFO[10:52:33] Done
Process 'Quant (Isobaric)' finished, exit code: 0 PhilosopherReport [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe report INFO[10:52:33] Executing Report v4.4.0
INFO[10:52:33] Creating reports
INFO[10:52:40] Done
Process 'PhilosopherReport' finished, exit code: 0 WorkspaceClean [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] X:\06_scripts\MSFragger_cmd\fragpipe\tools\philosopher_v4.4.0_windows_amd64\philosopher.exe workspace --clean --nocheck INFO[10:52:41] Executing Workspace v4.4.0
INFO[10:52:41] Removing workspace
INFO[10:52:41] Done
Process 'WorkspaceClean' finished, exit code: 0 TmtIntegrator [Work dir: X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4] java -Xmx24G -cp "X:\06_scripts\MSFragger_cmd\fragpipe\tools\tmt-integrator-3.4.jar" TMTIntegrator X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\tmt-integrator-conf.yml X:\01_users\Raphael\Vorversuche\Experiments\Lysate_test_TMT4\psm.tsv TMT-Integrator v3.4 UpdateColumns--- 0.05382 min. LoadPsms--- 0.00623 min. Take log and normalize--- 0.00000 min. PSM normalization--- 0.00000 min. outlierRemoval--- 0.00000 min. Collapse--- 0.00000 min. protNorm--- 0.00000 min. Report--- 0.00053 min. Finish!!! Process 'TmtIntegrator' finished, exit code: 0

Please cite: (Any searches) MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. Nat Methods 14:513 (2017) (Any searches) Fast deisotoping algorithm and its implementation in the MSFragger search engine. J. Proteome Res. 20:498 (2021) (Open search) Identification of modified peptides using localization-aware open search. Nat Commun. 11:4065 (2020) (Open search) Crystal-C: A Computational Tool for Refinement of Open Search Results. J. Proteome Res. 19.6:2511 (2020) (Open search) PTM-Shepherd: analysis and summarization of post-translational and chemical modifications from open search results. Mol Cell Proteomics 20:100018 (2020) (Glyco/labile search) Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco. Nat Methods 17:1125 (2020) (timsTOF PASEF) Fast quantitative analysis of timsTOF PASEF data with MSFragger and IonQuant. Mol Cell Proteomics 19:1575 (2020) (PSM validation with Percolator) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4:923 (2007) (Label-free quantification/SILAC) IonQuant Enables Accurate and Sensitive Label-Free Quantification With FDR-Controlled Match-Between-Runs. Mol Cell Proteomics 20:100077 (2021) (PeptideProphet/ProteinProphet/PTMProphet/Filtering) Philosopher: a versatile toolkit for shotgun proteomics data analysis. Nat Methods 17:869 (2020) (TMT-Integrator) Quantitative proteomic landscape of metaplastic breast carcinoma pathological subtypes and their relationship to triple-negative tumors. Nat Commun. 11:1723 (2020) (DIA-Umpire) DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics. Nat Methods 12:258 (2015) (DIA-NN) High sensitivity dia-PASEF proteomics with DIA-NN and FragPipe. bioRxiv doi:10.1101/2021.03.08.434385 (2021)

=============================================================ALL JOBS DONE IN 31.6 MINUTES=============================================================

prvst commented 2 years ago

Hi Frank, since it's related to the FragPipe headless mode, I'm moving the ticket to the FragPipe repository.

prvst commented 2 years ago

Here's what I gathered so far:

  1. Cannot be reproduced when running Philosopher standalone
  2. Cannot be reproduced when running Philosopher pipeline
  3. Cannot be reproduced when running FragPipe GUI
  4. The problem seems to happen only when running FragPipe headless with Percolator. When using PeptideProphet there's no problem.

@fstein is that it?

fstein commented 2 years ago

To No 4: The problem now also occurs when running PeptideProphet. When running Percolator, the problem was a little bit different. Then only the protein.tsv output seemed to be affected.

  1. The problem seems to be raw-file specific. At least I get this error with certain raw files only. With other raw files and the same settings, I did not observe this issue. With the raw file from the archive (https://oc.embl.de/index.php/s/9iEiAtMw6j0hSMK) I could reproduce the issue with the workflow file (which is also in the archive).
fstein commented 2 years ago

It's the same problem that is described in this post: https://github.com/Nesvilab/FragPipe/issues/747

fcyu commented 2 years ago

Hi Guys, thanks for your investigation. Following are my comments. I am also running @fstein's file to try to reproduce the bug.

Cannot be reproduced when running Philosopher standalone Cannot be reproduced when running FragPipe GUI The problem seems to happen only when running FragPipe headless with Percolator. When using PeptideProphet there's no problem.

If you look at how FragPipe GUI and headless modes run Philosopher, you will find that it runs the exactly the same command. The same command is also the one you will use when running Philosopher standalone. Thus, the results will be identical from FragPipe GUI, headless, and Philosopher standalone if you use the identical parameters and input. If you can show me that they are not, I would be very surprised and I will buy you a drink, seriously!

  1. The problem seems to be raw-file specific. At least I get this error with certain raw files only. With other raw files and the same settings, I did not observe this issue.

I guess the "raw-file" means mzML file right? Can you also upload the file without any issue?

It's the same problem that is described in this post: https://github.com/Nesvilab/FragPipe/issues/747

I have already confirmed that that issue is due to Philosopher. @prvst Can you take a look at that issue?

Thanks,

Fengchao

fcyu commented 2 years ago

Hi @prvst and @fstein ,

I reproduced the error with both PeptideProphet and Percolator. Other settings are the same. It seems that psm.tsv is not affected but ion.tsv, peptide.tsv, and protein.tsv are affected.

Following is the header from psm.tsv:

Spectrum    Spectrum File   Peptide Modified Peptide    Prev AA Next AA Peptide Length  Charge  Retention   Observed Mass   Calibrated Observed Mass    Observed M/Z    Calibrated Observed M/Z Calculated Peptide Mass Calculated M/Z  Delta Mass  Expectation Hyperscore  Nextscore   PeptideProphet Probability  Number of Enzymatic Termini Number of Missed Cleavages  Protein Start   Protein End Intensity   Assigned Modifications  Observed Modifications  Purity  Is Unique   Protein Protein ID  Entry Name  Gene    Protein Description Mapped Genes    Mapped Proteins Quan Usage  channel_126 channel_127N    channel_128C    channel_129N    channel_130C    channel_131N

Following is the header from ion.tsv:

Peptide Sequence    Modified Sequence   Prev AA Next AA Peptide Length  M/Z Charge  Observed Mass   Probability Expectation Spectral Count  Intensity   Assigned Modifications  Observed Modifications  Protein Protein ID  Entry Name  Gene    Protein Description Mapped Genes    Mapped Proteins channel_126126  channel_126127N channel_126128C channel_126129N channel_126130C channel_126131

I have uploaded all files to https://www.dropbox.com/sh/5qx1txol625khvg/AABlTbxLq4oDtYd7f0jaz4CNa?dl=0 . Please check the files in yufe_peptideprophet and yufe_percolator folders.

Best,

Fengchao

prvst commented 2 years ago

Did you use the headless mode or the GUI?

fcyu commented 2 years ago

GUI. And again, they are really the same because they are using the same codebase and running the same command.

FragPipe itself won't have anything to do with the bug because the files are generated by Philosopher. FragPipe just run the command.

prvst commented 2 years ago

@fstein are you using the updated RC? which one are you using it?

fstein commented 2 years ago

Great, that someone could finally reproduce this issue :-) I am using the latest philosopher version 4.4.0. The 4.5.1-RC6 did not give me any TMT quantification at all. All files (except the psm.tsv) were missing the channel_126... columns.

fstein commented 2 years ago

I could also reproduce this issue using the GUI.

fstein commented 2 years ago

@prvst The mzML file is also part of the download link: https://oc.embl.de/index.php/s/9iEiAtMw6j0hSMK

prvst commented 2 years ago

I could also reproduce this issue using the GUI.

It seems a little different from the case we were talking via email, but relatable, yes. You grabbed the version before I did the reporting. I'll send you the latest one. I ran your data here and got all headers and tables just fine.

fcyu commented 2 years ago

Thanks Felipe @prvst , can you also put the fixed version here so that we all can test?

Thanks,

Fengchao

fstein commented 2 years ago

Happy to test the new version as well :-)

fstein commented 2 years ago

With philosopher_v4.5.1-RC8 the issue seems to be resolved :-)

fstein commented 2 years ago

Here is the log-file from this run: 21:47:25 WARN : The output directory is not empty. Some files might be overwritten in: C:\MS_Testrun_Raphael_126126

System OS: Windows 10, Architecture: AMD64 Java Info: 11.0.15, OpenJDK 64-Bit Server VM, Eclipse Adoptium

Version info: FragPipe version 18.0 MSFragger version 3.5 Philosopher version 4.5.1-RC8

LCMS files: Experiment/Group:

16 commands to execute: CheckCentroid C:\Fragpipe\fragpipe\jre\bin\java.exe -Xmx46G -cp "C:\Fragpipe\fragpipe\lib\fragpipe-18.0.jar;C:\Fragpipe\fragpipe\tools\batmass-io-1.25.5.jar" com.dmtavt.fragpipe.util.CheckCentroid C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML 15 WorkspaceCleanInit [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --init --nocheck --temp C:\Users\fstein\AppData\Local\Temp\caa3fd62-4719-404e-9485-cfc01cf11679 MSFragger [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx46G C:\Fragpipe\fragpipe\tools\MSFragger-3.5\MSFragger-3.5.jar C:\MS_Testrun_Raphael_126126\fragger.params C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML PeptideProphet: Workspace init [Work dir: C:\MS_Testrun_Raphael_126126\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --init --nocheck --temp C:\Users\fstein\AppData\Local\Temp\32c6dc2b-35f6-4f79-9daf-581facc8ee95 PeptideProphet [Work dir: C:\MS_Testrun_Raphael_126126\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] C:\Fragpipe\fragpipe\tools\philosopherv4.5.1-RC8\philosopher-v4.5.1-RC8.exe peptideprophet --decoyprobs --ppm --accmass --nonparam --expectscore --decoy rev --database C:\MS_Testrun_Raphael_126126\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas ..\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML PeptideProphet: Delete temp C:\Fragpipe\fragpipe\jre\bin\java.exe -cp C:\Fragpipe\fragpipe\lib\fragpipe-18.0.jar com.github.chhh.utils.FileDelete C:\MS_Testrun_Raphael_126126\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp Rewrite pepxml [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\jre\bin\java.exe -cp C:\Fragpipe\fragpipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML ProteinProphet [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined C:\MS_Testrun_Raphael_126126\filelist_proteinprophet.txt PhilosopherDbAnnotate [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe database --annotate C:\MS_Testrun_Raphael_126126\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries05082022.fasta.fas --prefix rev PhilosopherFilter [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopherv4.5.1-RC8\philosopher-v4.5.1-RC8.exe filter --sequential --picked --prot 0.01 --tag rev --pepxml C:\MS_Testrun_Raphael_126126 --protxml C:\MS_Testrun_Raphael_126126\combined.prot.xml --razor FreeQuant [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe freequant --ptw 0.4 --tol 10 --dir C:\MS_Testrun_Raphael_126126 Quant (Isobaric) [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe labelquant --tol 20 --level 2 --plex 6 --annot C:\MS_Testrun_Raphael_126126\annotation.txt --brand tmt --dir C:\MS_Testrun_Raphael_126126 PhilosopherReport [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe report WorkspaceClean [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --clean --nocheck TmtIntegrator [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\jre\bin\java.exe -Xmx46G -cp "C:\Fragpipe\fragpipe\tools\tmt-integrator-3.3.3.jar" TMTIntegrator C:\MS_Testrun_Raphael_126126\tmt-integrator-conf.yml C:\MS_Testrun_Raphael_126126\psm.tsv


Execution order:

    Cmd: [START], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [CheckCentroid], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [WorkspaceCleanInit], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [MSFragger], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [PeptideProphet], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [ProteinProphet], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [PhilosopherDbAnnotate], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [PhilosopherFilter], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [FreeQuant], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [Quant (Isobaric)], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [PhilosopherReport], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [WorkspaceClean], Work dir: [C:\MS_Testrun_Raphael_126126]
    Cmd: [TmtIntegrator], Work dir: [C:\MS_Testrun_Raphael_126126]

>rev_sp|A0A024B7W1|POLG_ZIKVF Genome polyprotein OS=Zika virus (isolate ZIKV/Human/French Polynesia/10087PF/2013) OX=2043570 PE=1 SV=1
>rev_sp|O75335-1|LIPA4_HUMAN Isoform 1 of Liprin-alpha-4 OS=Homo sapiens OX=9606 GN=PPFIA4
>rev_sp|P11049-3|CD37_HUMAN Isoform 3 of Leukocyte antigen CD37 OS=Homo sapiens OX=9606 GN=CD37
>rev_sp|P41229-5|KDM5C_HUMAN Isoform 5 of Lysine-specific demethylase 5C OS=Homo sapiens OX=9606 GN=KDM5C
>rev_sp|Q12792-4|TWF1_HUMAN Isoform 4 of Twinfilin-1 OS=Homo sapiens OX=9606 GN=TWF1
>rev_sp|Q5SWX8|ODR4_HUMAN Protein odr-4 homolog OS=Homo sapiens OX=9606 GN=ODR4 PE=1 SV=1
>rev_sp|Q7Z6J4-5|FGD2_HUMAN Isoform 4 of FYVE, RhoGEF and PH domain-containing protein 2 OS=Homo sapiens OX=9606 GN=FGD2
>rev_sp|Q8TAK5|GABP2_HUMAN GA-binding protein subunit beta-2 OS=Homo sapiens OX=9606 GN=GABPB2 PE=1 SV=1
>rev_sp|Q99523|SORT_HUMAN Sortilin OS=Homo sapiens OX=9606 GN=SORT1 PE=1 SV=3
>rev_sp|Q9NTQ9|CXB4_HUMAN Gap junction beta-4 protein OS=Homo sapiens OX=9606 GN=GJB4 PE=1 SV=1
>rev_sp|V5IVB1|L_BCCV RNA-directed RNA polymerase L OS=Black Creek Canal orthohantavirus OX=1980460 GN=RdRp PE=3 SV=1
>sp|O75334-2|LIPA2_HUMAN Isoform 2 of Liprin-alpha-2 OS=Homo sapiens OX=9606 GN=PPFIA2
>sp|P10966-7|CD8B_HUMAN Isoform 6 of T-cell surface glycoprotein CD8 beta chain OS=Homo sapiens OX=9606 GN=CD8B
>sp|P41223|BUD31_HUMAN Protein BUD31 homolog OS=Homo sapiens OX=9606 GN=BUD31 PE=1 SV=2
>sp|Q12791-2|KCMA1_HUMAN Isoform 2 of Calcium-activated potassium channel subunit alpha-1 OS=Homo sapiens OX=9606 GN=KCNMA1
>sp|Q5SWA1|PR15B_HUMAN Protein phosphatase 1 regulatory subunit 15B OS=Homo sapiens OX=9606 GN=PPP1R15B PE=1 SV=1
>sp|Q7Z6I8-2|CE024_HUMAN Isoform 2 of UPF0461 protein C5orf24 OS=Homo sapiens OX=9606 GN=C5orf24
>sp|Q8TAG5|VTM2A_HUMAN V-set and transmembrane domain-containing protein 2A OS=Homo sapiens OX=9606 GN=VSTM2A PE=1 SV=3
>sp|Q99504-2|EYA3_HUMAN Isoform 2 of Eyes absent homolog 3 OS=Homo sapiens OX=9606 GN=EYA3
>sp|Q9NTK5-3|OLA1_HUMAN Isoform 3 of Obg-like ATPase 1 OS=Homo sapiens OX=9606 GN=OLA1
>sp|Q9YX78|PB2_I97A0 Polymerase basic protein 2 (Fragment) OS=Influenza A virus (strain A/Chicken/Hong Kong/220/1997 H5N1 genotype Gs/Gd) OX=100834 GN=PB2 PE=3 SV=1
C:\MS_Testrun_Raphael_126126\annotation.txt:
126 channel_126
127N channel_127N
128C channel_128C
129N channel_129N
130C channel_130C
131N channel_131N
# FragPipe v18.0ui state cache

# Please edit the following path to point to the correct location.
# In Windows, please replace single '\' with '\\'
database.db-path=C\:\\MS_Testrun_Raphael_126126\\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas

crystalc.run-crystalc=false
database.decoy-tag=rev_
diann.fragpipe.cmd-opts=
diann.library=
diann.q-value=0.01
diann.quantification-strategy=3
diann.run-dia-nn=false
diann.run-specific-protein-q-value=0.01
diann.unrelated-runs=false
diann.use-predicted-spectra=false
diaumpire.AdjustFragIntensity=true
diaumpire.BoostComplementaryIon=false
diaumpire.CorrThreshold=0
diaumpire.DeltaApex=0.2
diaumpire.ExportPrecursorPeak=false
diaumpire.Q1=true
diaumpire.Q2=true
diaumpire.Q3=true
diaumpire.RFmax=500
diaumpire.RPmax=25
diaumpire.RTOverlap=0.3
diaumpire.SE.EstimateBG=false
diaumpire.SE.IsoPattern=0.3
diaumpire.SE.MS1PPM=10
diaumpire.SE.MS2PPM=20
diaumpire.SE.MS2SN=1.1
diaumpire.SE.MassDefectFilter=true
diaumpire.SE.MassDefectOffset=0.1
diaumpire.SE.NoMissedScan=1
diaumpire.SE.SN=1.1
diaumpire.run-diaumpire=false
fragpipe-config.bin-ionquant=C\:\\Fragpipe\\fragpipe\\tools\\IonQuant-1.8.0.jar
fragpipe-config.bin-msfragger=C\:\\Fragpipe\\fragpipe\\tools\\MSFragger-3.5\\MSFragger-3.5.jar
fragpipe-config.bin-philosopher=C\:\\Fragpipe\\fragpipe\\tools\\philosopher_v4.5.1-RC8\\philosopher-v4.5.1-RC8.exe
fragpipe-config.bin-python=C\:\\Users\\fstein\\AppData\\Local\\Programs\\Python\\Python310\\python
freequant.mz-tol=10
freequant.rt-tol=0.4
freequant.run-freequant=false
ionquant.excludemods=
ionquant.heavy=
ionquant.ibaq=0
ionquant.imtol=0.05
ionquant.ionfdr=0.01
ionquant.light=
ionquant.locprob=0.75
ionquant.maxlfq=1
ionquant.mbr=0
ionquant.mbrimtol=0.05
ionquant.mbrmincorr=0
ionquant.mbrrttol=1
ionquant.mbrtoprun=100000
ionquant.medium=
ionquant.minexps=1
ionquant.minfreq=0
ionquant.minions=2
ionquant.minisotopes=2
ionquant.minscans=3
ionquant.mztol=10
ionquant.normalization=1
ionquant.peptidefdr=1
ionquant.proteinfdr=1
ionquant.proteinquant=2
ionquant.requantify=1
ionquant.rttol=0.4
ionquant.run-ionquant=true
ionquant.tp=0
ionquant.uniqueness=0
ionquant.use-labeling=false
ionquant.use-lfq=true
ionquant.writeindex=0
msbooster.predict-rt=true
msbooster.predict-spectra=true
msbooster.run-msbooster=false
msbooster.use-correlated-features=false
msfragger.Y_type_masses=
msfragger.activation_types=all
msfragger.add_topN_complementary=0
msfragger.allowed_missed_cleavage_1=2
msfragger.allowed_missed_cleavage_2=2
msfragger.calibrate_mass=2
msfragger.check_spectral_files=true
msfragger.clip_nTerm_M=true
msfragger.data_type=0
msfragger.deisotope=1
msfragger.delta_mass_exclude_ranges=(-1.5,3.5)
msfragger.deneutralloss=1
msfragger.diagnostic_fragments=
msfragger.diagnostic_intensity_filter=0
msfragger.digest_max_length=50
msfragger.digest_min_length=7
msfragger.fragment_ion_series=b,y
msfragger.fragment_mass_tolerance=20
msfragger.fragment_mass_units=1
msfragger.intensity_transform=0
msfragger.ion_series_definitions=
msfragger.isotope_error=-1/0/1/2/3
msfragger.labile_search_mode=off
msfragger.localize_delta_mass=false
msfragger.mass_diff_to_variable_mod=0
msfragger.mass_offsets=0
msfragger.max_fragment_charge=2
msfragger.max_variable_mods_combinations=5000
msfragger.max_variable_mods_per_peptide=3
msfragger.min_fragments_modelling=2
msfragger.min_matched_fragments=4
msfragger.min_sequence_matches=2
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.01
msfragger.misc.fragger.clear-mz-hi=131.5
msfragger.misc.fragger.clear-mz-lo=125.5
msfragger.misc.fragger.digest-mass-hi=5000
msfragger.misc.fragger.digest-mass-lo=200
msfragger.misc.fragger.enzyme-dropdown-1=trypsin
msfragger.misc.fragger.enzyme-dropdown-2=null
msfragger.misc.fragger.precursor-charge-hi=6
msfragger.misc.fragger.precursor-charge-lo=1
msfragger.misc.fragger.remove-precursor-range-hi=1.5
msfragger.misc.fragger.remove-precursor-range-lo=-1.5
msfragger.misc.slice-db=1
msfragger.num_enzyme_termini=2
msfragger.output_format=pepXML_pin
msfragger.output_max_expect=50
msfragger.output_report_topN=1
msfragger.override_charge=false
msfragger.precursor_mass_lower=-20
msfragger.precursor_mass_mode=selected
msfragger.precursor_mass_units=1
msfragger.precursor_mass_upper=20
msfragger.precursor_true_tolerance=20
msfragger.precursor_true_units=1
msfragger.remainder_fragment_masses=
msfragger.remove_precursor_peak=1
msfragger.report_alternative_proteins=true
msfragger.restrict_deltamass_to=all
msfragger.run-msfragger=true
msfragger.search_enzyme_cut_1=KR
msfragger.search_enzyme_cut_2=
msfragger.search_enzyme_name=stricttrypsin
msfragger.search_enzyme_name_1=trypsin
msfragger.search_enzyme_name_2=null
msfragger.search_enzyme_nocut_1=P
msfragger.search_enzyme_nocut_2=
msfragger.search_enzyme_sense_1=C
msfragger.search_enzyme_sense_2=C
msfragger.table.fix-mods=0.000000,C-Term Peptide,true,-1; 0.000000,N-Term Peptide,true,-1; 0.000000,C-Term Protein,true,-1; 0.000000,N-Term Protein,true,-1; 0.000000,G (glycine),true,-1; 0.000000,A (alanine),true,-1; 0.000000,S (serine),true,-1; 0.000000,P (proline),true,-1; 0.000000,V (valine),true,-1; 0.000000,T (threonine),true,-1; 57.021460,C (cysteine),true,-1; 0.000000,L (leucine),true,-1; 0.000000,I (isoleucine),true,-1; 0.000000,N (asparagine),true,-1; 0.000000,D (aspartic acid),true,-1; 0.000000,Q (glutamine),true,-1; 229.162932,K (lysine),true,-1; 0.000000,E (glutamic acid),true,-1; 0.000000,M (methionine),true,-1; 0.000000,H (histidine),true,-1; 0.000000,F (phenylalanine),true,-1; 0.000000,R (arginine),true,-1; 0.000000,Y (tyrosine),true,-1; 0.000000,W (tryptophan),true,-1; 0.000000,B ,true,-1; 0.000000,J,true,-1; 0.000000,O,true,-1; 0.000000,U,true,-1; 0.000000,X,true,-1; 0.000000,Z,true,-1
msfragger.table.var-mods=15.994900,M,true,3; 42.010600,[^,true,1; 229.162932,n^,true,1
msfragger.track_zero_topN=0
msfragger.use_all_mods_in_first_search=false
msfragger.use_topN_peaks=300
msfragger.write_calibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=true
percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc
percolator.keep-tsv-files=false
percolator.min-prob=0.5
percolator.run-percolator=false
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--sequential --picked --prot 0.01
phi-report.pep-level-summary=false
phi-report.philosoher-msstats=false
phi-report.print-decoys=false
phi-report.prot-level-summary=false
phi-report.run-report=true
protein-prophet.cmd-opts=--maxppmdiff 2000000 --minprob 0.5
protein-prophet.run-protein-prophet=true
ptmprophet.cmdline=
ptmprophet.run-ptmprophet=false
ptmshepherd.adv_params=false
ptmshepherd.annotation-common=false
ptmshepherd.annotation-custom=false
ptmshepherd.annotation-glyco=false
ptmshepherd.annotation-unimod=true
ptmshepherd.annotation_file=
ptmshepherd.annotation_tol=0.01
ptmshepherd.assign_glycans=true
ptmshepherd.cap_y_ions=
ptmshepherd.decoy_type=1
ptmshepherd.diag_ions=
ptmshepherd.diagextract_mode=false
ptmshepherd.diagmine_diagMinFoldChange=3.0
ptmshepherd.diagmine_diagMinSpecDiff=00.2
ptmshepherd.diagmine_fragMinFoldChange=3.0
ptmshepherd.diagmine_fragMinPropensity=00.1
ptmshepherd.diagmine_fragMinSpecDiff=00.1
ptmshepherd.diagmine_minIons=25
ptmshepherd.diagmine_minIonsPerSpec=2
ptmshepherd.diagmine_minPeps=25
ptmshepherd.diagmine_minSpecDiff=0.25
ptmshepherd.diagmine_mode=false
ptmshepherd.diagmine_pepMinFoldChange=3.0
ptmshepherd.diagmine_pepMinSpecDiff=00.2
ptmshepherd.glyco_adducts=
ptmshepherd.glyco_fdr=1.00
ptmshepherd.glyco_isotope_max=3
ptmshepherd.glyco_isotope_min=-1
ptmshepherd.glyco_mode=false
ptmshepherd.glyco_ppm_tol=50
ptmshepherd.glycodatabase=
ptmshepherd.histo_smoothbins=2
ptmshepherd.iontype_a=false
ptmshepherd.iontype_b=true
ptmshepherd.iontype_c=true
ptmshepherd.iontype_x=false
ptmshepherd.iontype_y=true
ptmshepherd.iontype_z=true
ptmshepherd.localization_allowed_res=
ptmshepherd.localization_background=4
ptmshepherd.max_adducts=0
ptmshepherd.n_glyco=true
ptmshepherd.normalization-psms=true
ptmshepherd.normalization-scans=false
ptmshepherd.output_extended=false
ptmshepherd.peakpicking_mass_units=0
ptmshepherd.peakpicking_minPsm=10
ptmshepherd.peakpicking_promRatio=0.3
ptmshepherd.peakpicking_width=0.002
ptmshepherd.precursor_mass_units=0
ptmshepherd.precursor_tol=0.01
ptmshepherd.print_decoys=false
ptmshepherd.prob_dhexOx=
ptmshepherd.prob_dhexY=
ptmshepherd.prob_neuacOx=
ptmshepherd.prob_neugcOx=
ptmshepherd.prob_phosphoOx=
ptmshepherd.prob_regY=
ptmshepherd.prob_sulfoOx=
ptmshepherd.remainder_masses=
ptmshepherd.remove_glycan_delta_mass=true
ptmshepherd.run-shepherd=false
ptmshepherd.spectra_maxfragcharge=2
ptmshepherd.spectra_ppmtol=20
ptmshepherd.varmod_masses=
quantitation.run-label-free-quant=false
run-psm-validation=true
run-validation-tab=true
speclibgen.easypqp.extras.max_delta_ppm=15
speclibgen.easypqp.extras.max_delta_unimod=0.02
speclibgen.easypqp.extras.rt_lowess_fraction=0
speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM
speclibgen.easypqp.rt-cal=ciRT
speclibgen.easypqp.select-file.text=
speclibgen.easypqp.select-im-file.text=
speclibgen.keep-intermediate-files=false
speclibgen.run-speclibgen=false
speclibgen.use-easypqp=false
speclibgen.use-spectrast=true
tmtintegrator.add_Ref=1
tmtintegrator.aggregation_method=1
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.glyco_qval=-1
tmtintegrator.groupby=0
tmtintegrator.max_pep_prob_thres=0.9
tmtintegrator.min_ntt=0
tmtintegrator.min_pep_prob=0.9
tmtintegrator.min_percent=0.05
tmtintegrator.min_purity=0.5
tmtintegrator.min_site_prob=-1
tmtintegrator.mod_tag=none
tmtintegrator.ms1_int=true
tmtintegrator.outlier_removal=true
tmtintegrator.print_RefInt=false
tmtintegrator.prot_exclude=none
tmtintegrator.prot_norm=0
tmtintegrator.psm_norm=false
tmtintegrator.quant_level=2
tmtintegrator.ref_tag=channel_126
tmtintegrator.run-tmtintegrator=true
tmtintegrator.tolerance=20
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=1
tmtintegrator.unique_pep=true
tmtintegrator.use_glycan_composition=false
workdir=C\:\\MS_Testrun_Raphael_126126
workflow.input.data-type.im-ms=false
workflow.input.data-type.regular-ms=true
workflow.ram=0
workflow.threads=15

CheckCentroid C:\Fragpipe\fragpipe\jre\bin\java.exe -Xmx46G -cp "C:\Fragpipe\fragpipe\lib\fragpipe-18.0.jar;C:\Fragpipe\fragpipe\tools\batmass-io-1.25.5.jar" com.dmtavt.fragpipe.util.CheckCentroid C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML 15 Done in 1699 ms. Process 'CheckCentroid' finished, exit code: 0 WorkspaceCleanInit [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --clean --nocheck INFO[21:47:27] Executing Workspace v4.5.1
INFO[21:47:27] Removing workspace
INFO[21:47:27] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --init --nocheck --temp C:\Users\fstein\AppData\Local\Temp\caa3fd62-4719-404e-9485-cfc01cf11679 INFO[21:47:27] Executing Workspace v4.5.1
INFO[21:47:27] Creating workspace
INFO[21:47:27] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx46G C:\Fragpipe\fragpipe\tools\MSFragger-3.5\MSFragger-3.5.jar C:\MS_Testrun_Raphael_126126\fragger.params C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML MSFragger version MSFragger-3.5 Batmass-IO version 1.25.5 timsdata library version timsdata-2-8-7-1 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. System OS: Windows 10, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK JVM started with 46 GB memory Checking database... Checking spectral files... C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML: Scans = 46464 ***FIRST SEARCH**** Parameters: num_threads = 15 database_name = C:\MS_Testrun_Raphael_126126\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 20.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mgf = 0 isotope_error = 0/1 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = trypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = P allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = pepxml_pin output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = false override_charge = false precursor_charge_low = 1 precursor_charge_high = 6 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 200.0 digest_mass_range_high = 5000.0 max_fragment_charge = 1 deisotope = 1 deneutralloss = true track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 300 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.01 intensity_transform = 0 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 125.5 clear_mz_range_high = 131.5 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.994900 M 3 variable_mod_02 = 42.010600 [^ 1 variable_mod_03 = 229.162932 n^ 1 add_A_alanine = 0.000000 add_B_user_amino_acid = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_J_user_amino_acid = 0.000000 add_K_lysine = 229.162932 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.000000 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_U_user_amino_acid = 0.000000 # U = selenocysteine (150.95363 Da) add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_X_user_amino_acid = 0.000000 add_Y_tyrosine = 0.000000 add_Z_user_amino_acid = 0.000000 Number of unique peptides of length 7: 303876 of length 8: 296726 of length 9: 292420 of length 10: 281062 of length 11: 269493 of length 12: 258141 of length 13: 253380 of length 14: 237594 of length 15: 228730 of length 16: 214992 of length 17: 203000 of length 18: 196793 of length 19: 191186 of length 20: 175550 of length 21: 168567 of length 22: 159513 of length 23: 149507 of length 24: 143684 of length 25: 135137 of length 26: 127245 of length 27: 121805 of length 28: 118524 of length 29: 107768 of length 30: 102277 of length 31: 96334 of length 32: 91835 of length 33: 86629 of length 34: 80444 of length 35: 74852 of length 36: 72825 of length 37: 67042 of length 38: 62832 of length 39: 57539 of length 40: 54776 of length 41: 51558 of length 42: 46779 of length 43: 40730 of length 44: 32663 of length 45: 24640 of length 46: 16363 of length 47: 9945 of length 48: 5681 of length 49: 2940 of length 50: 1619 In total 5714996 peptides. Generated 17707829 modified peptides. Number of peptides with more than 5000 modification patterns: 0 Selected fragment index width 0.10 Da. 744971344 fragments to be searched in 1 slices (11.10 GB total) Operating on slice 1 of 1: Fragment index slice generated in 8.36 s

  1. Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML 2.4 s | deisotoping 0.8 s [progress: 46148/46148 (100%) - 9954 spectra/s] 4.6s | postprocessing 0.3 s ***FIRST SEARCH DONE IN 0.579 MIN**
**MASS CALIBRATION AND PARAMETER OPTIMIZATION ----- --------------- --------------- --------------- --------------- MS1 (Old) MS1 (New) MS2 (Old) MS2 (New)
Run Median MAD Median MAD Median MAD Median MAD
001 0.37 1.25 0.03 1.04 -1.31 1.14 -0.09 1.12
----- --------------- --------------- --------------- ---------------
Finding the optimal parameters: ------- ------- ------- ------- ------- ------- ------- ------- ------- MS2 5 7 10 15 20 25 30 50
Count 30534 30649 30739 30711 skip rest
------- ------- ------- ------- ------- ------- ------- ------- -------
------- ------- ------- ------- ------- ------- -------
Peaks 300_0 200_0 175_0 150_1 125_1 100_1
------- ------- ------- ------- ------- ------- -------
Count 30771 30767 skip rest
------- ------- ------- ------- ------- ------- -------
------- -------
Int. 1
------- -------
Count 30897
------- -------
------- -------
Rm P. 0
------- -------
Count 30850
------- -------

New fragment_mass_tolerance = 10 PPM New use_topN_peaks = 300 New minimum_ratio = 0.000000 New intensity_transform = 1 New remove_precursor_peak = 1 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 2.037 MIN*****

****MAIN SEARCH**** Checking database... Parameters: num_threads = 15 database_name = C:\MS_Testrun_Raphael_126126\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 10.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mgf = 0 isotope_error = -1/0/1/2/3 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = trypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = P allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = pepxml_pin output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = true override_charge = false precursor_charge_low = 1 precursor_charge_high = 6 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 200.0 digest_mass_range_high = 5000.0 max_fragment_charge = 1 deisotope = 1 deneutralloss = true track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 300 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.0 intensity_transform = 1 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 125.5 clear_mz_range_high = 131.5 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.994900 M 3 variable_mod_02 = 42.010600 [^ 1 variable_mod_03 = 229.162932 n^ 1 add_A_alanine = 0.000000 add_B_user_amino_acid = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_J_user_amino_acid = 0.000000 add_K_lysine = 229.162932 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.000000 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_U_user_amino_acid = 0.000000 # U = selenocysteine (150.95363 Da) add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_X_user_amino_acid = 0.000000 add_Y_tyrosine = 0.000000 add_Z_user_amino_acid = 0.000000 Number of unique peptides of length 7: 303876 of length 8: 296726 of length 9: 292420 of length 10: 281062 of length 11: 269493 of length 12: 258141 of length 13: 253380 of length 14: 237594 of length 15: 228730 of length 16: 214992 of length 17: 203000 of length 18: 196793 of length 19: 191186 of length 20: 175550 of length 21: 168567 of length 22: 159513 of length 23: 149507 of length 24: 143684 of length 25: 135137 of length 26: 127245 of length 27: 121805 of length 28: 118524 of length 29: 107768 of length 30: 102277 of length 31: 96334 of length 32: 91835 of length 33: 86629 of length 34: 80444 of length 35: 74852 of length 36: 72825 of length 37: 67042 of length 38: 62832 of length 39: 57539 of length 40: 54776 of length 41: 51558 of length 42: 46779 of length 43: 40730 of length 44: 32663 of length 45: 24640 of length 46: 16363 of length 47: 9945 of length 48: 5681 of length 49: 2940 of length 50: 1619 In total 5714996 peptides. Generated 17707829 modified peptides. Number of peptides with more than 5000 modification patterns: 0 Selected fragment index width 0.05 Da. 744971344 fragments to be searched in 1 slices (11.10 GB total) Operating on slice 1 of 1: Fragment index slice generated in 7.78 s

  1. Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzBIN_calibrated 0.4 s [progress: 45984/45984 (100%) - 8312 spectra/s] 5.5s | ramaping alternative proteins and postprocessing 8.1 s Process 'MSFragger' finished, exit code: 0 MAIN SEARCH DONE IN 0.572 MIN

***TOTAL TIME 3.188 MIN**** PeptideProphet: Workspace init [Work dir: C:\MS_Testrun_Raphael_126126\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --init --nocheck --temp C:\Users\fstein\AppData\Local\Temp\32c6dc2b-35f6-4f79-9daf-581facc8ee95 INFO[21:50:39] Executing Workspace v4.5.1
INFO[21:50:39] Creating workspace
INFO[21:50:39] Done
Process 'PeptideProphet: Workspace init' finished, exit code: 0 PeptideProphet [Work dir: C:\MS_Testrun_Raphael_126126\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp] C:\Fragpipe\fragpipe\tools\philosopherv4.5.1-RC8\philosopher-v4.5.1-RC8.exe peptideprophet --decoyprobs --ppm --accmass --nonparam --expectscore --decoy rev --database C:\MS_Testrun_Raphael_126126\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas ..\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML INFO[21:50:40] Executing PeptideProphet v4.5.1
file 1: C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML processed altogether 44060 results INFO: Results written to file: C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml

using Accurate Mass Bins using PPM mass difference Using Decoy Label "rev_". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling) adding ACCMASS mixture distribution using search_offsets in ACCMASS mixture distr: 0 init with X! Tandem trypsin PeptideProphet (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB read in 0 1+, 26053 2+, 13596 3+, 4411 4+, 0 5+, 0 6+, and 0 7+ spectra. Found 5059 Decoys, and 39001 Non-Decoys MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... Initialising statistical models ... Iterations: .........10.........20.....WARNING: Mixture model quality test failed for charge (1+).

WARNING: Mixture model quality test failed for charge (5+). WARNING: Mixture model quality test failed for charge (6+). WARNING: Mixture model quality test failed for charge (7+). model complete after 26 iterations Process 'PeptideProphet' finished, exit code: 0 PeptideProphet: Delete temp INFO[21:54:52] Done
C:\Fragpipe\fragpipe\jre\bin\java.exe -cp C:\Fragpipe\fragpipe\lib\fragpipe-18.0.jar com.github.chhh.utils.FileDelete C:\MS_Testrun_Raphael_126126\fragpipe-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pepXML-temp Process 'PeptideProphet: Delete temp' finished, exit code: 0 Rewrite pepxml [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\jre\bin\java.exe -cp C:\Fragpipe\fragpipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml C:\MS_Testrun_Raphael_126126\Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.mzML Fixing pepxml: C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml Writing output to: C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml10421194003680497064.temp-rewrite Deleting file: C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml Moving rewritten file to original location: [C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml10421194003680497064.temp-rewrite] -> [C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml] Process 'Rewrite pepxml' finished, exit code: 0 ProteinProphet [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined C:\MS_Testrun_Raphael_126126\filelist_proteinprophet.txt INFO[21:54:54] Executing ProteinProphet v4.5.1
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64)) (no FPKM) (no groups) (using degen pep info) Reading in C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml... ...read in 0 1+, 19911 2+, 10341 3+, 2304 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.5

Initializing 28047 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating protein lengths and molecular weights from database c:/MS_Testrun_Raphael_126126/2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries_05082022.fasta.fas .........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........14000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........15000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........16000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........17000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........18000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........19000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........20000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........21000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........22000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........23000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........24000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........25000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........26000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........27000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........28000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........29000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........30000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........31000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........32000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........33000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........34000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........35000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........36000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........37000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........38000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........39000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........40000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........41000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........42000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........43000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........44000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........45000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........46000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........47000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........48000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........49000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........50000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........51000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........52000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........53000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........54000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........55000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........56000 .........:.........:.........:.........:.........:.........:.........:.........:.........:WARNING: Found the following zero-mass residues in protein entry sp|P07203|GPX1_HUMAN : U MCAARLAAAAAAAQSVYAFSARPLAGGEPVSLGSLRGKVLLIENVASLUGTTVRDYTQMNELQRRLGPRGLVVLGFPCNQFGHQENAKNEEILNSLKYVRPGGGFEPNFMLFEKCEVNGAGAHPLFAFLREALPAPSDDATALMTDPKLITWSPVCRNDVAWNFEKFLVGPDGVPLRRYSRRFQTIDIEPDIEALLSQGPSCA .........57000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........58000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........59000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........60000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........61000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........62000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........63000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........64000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........65000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........66000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........67000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........68000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........69000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........70000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........71000 .........:.........:.....WARNING: Found the following zero-mass residues in protein entry sp|Q16881-2|TRXR1_HUMAN : U MLSRLVLNSWAQAIIRPRPPKVLGLQVTTFSEAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-3|TRXR1_HUMAN : U MQQVMLTCKGVNRGHAVPAGPGRKPRPRRSSRLLAGEKHLTRSALLLCHTEDGRALEGTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-4|TRXR1_HUMAN : U MSCEDGRALEGTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG .WARNING: Found the following zero-mass residues in protein entry sp|Q16881-5|TRXR1_HUMAN : U MNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-6|TRXR1_HUMAN : U MPVDDYWLCLPASCARPFVQTVRVVQSCPHCCWFPGVLPSVPEPLRMPAMLPTGSHSAVLPPSHCSTAPPSTSQEPSSSADPKLCLSPPTSDSRQERNVQFGLAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881-7|TRXR1_HUMAN : U MVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q16881|TRXR1_HUMAN : U MGCAEGKAVAAAAPTELQTKGKNGDGRRRSAKDHHPGKTLPENPAGFTSTATADSRALLQAYIDGHSVVIFSRSTCTRCTEVKKLFKSLCVPYFVLELDQTEDGRALEGTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCUG ...:.........:.........:.........:.........:.........:.........:.........72000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........73000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........74000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........75000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........76000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........77000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........78000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........79000 .........:........WARNING: Found the following zero-mass residues in protein entry sp|Q86VQ6|TRXR3_HUMAN : U MERSPPQSPGPGKAGDAPNRRSGHVRGARVLSPPGRRARLSSPGPSRSSEAREELRRHLVGLIERSRVVIFSKSYCPHSTRVKELFSSLGVECNVLELDQVDDGARVQEVLSEITNQKTVPNIFVNKVHVGGCDQTFQAYQSGLLQKLLQEDLAYDYDLIIIGGGSGGLSCAKEAAILGKKVMVLDFVVPSPQGTSWGLGGTCVNVGCIPKKLMHQAALLGQALCDSRKFGWEYNQQVRHNWETMTKAIQNHISSLNWGYRLSLREKAVAYVNSYGEFVEHHKIKATNKKGQETYYTAAQFVIATGERPRYLGIQGDKEYCITSDDLFSLPYCPGKTLVVGASYVALECAGFLAGFGLDVTVMVRSILLRGFDQEMAEKVGSYMEQHGVKFLRKFIPVMVQQLEKGSPGKLKVLAKSTEGTETIEGVYNTVLLAIGRDSCTRKIGLEKIGVKINEKSGKIPVNDVEQTNVPYVYAVGDILEDKPELTPVAIQSGKLLAQRLFGASLEKCDYINVPTTVFTPLEYGCCGLSEEKAIEVYKKENLEIYHTLFWPLEWTVAGRENNTCYAKIICNKFDHDRVIGFHILGPNAGEVTQGFAAAMKCGLTKQLLDDTIGIHPTCGEVFTTLEITKSSGLDITQKGCUG .:.........:.........:.........:.........:.........:.........:.........:.........80000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........81000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........82000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........83000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........84000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........85000 ........WARNING: Found the following zero-mass residues in protein entry sp|Q92813-2|IOD2_HUMAN : UU MGILSVDLLITLQILPVFFSNCLFLALYDSVILLKHVVLLLSRSKSTRGEWRRMLTSEGLRCVWKSFLLDAYKQLNCPPSGFSKDGHILCLVYEAYKSRLLVYSHLDLWMVKLGEDAPNSSVVHVSSTEGGDNSGNGTQEKIAEGATCHLLDFASPERPLVVNFGSATUPPFTSQLPAFRKLVEEFSSVADFLLVYIDEAHPSDGWAIPGDSSLSFEVKKHQNQEDRCAAAQQLLERFSLPPQCRVVADRMDNNANIAYGVAFERVCIVQRQKIAYLGGKGPFSYNLQEVRHWLEKNFSKRUKKTRLAG WARNING: Found the following zero-mass residues in protein entry sp|Q92813|IOD2_HUMAN : UU MGILSVDLLITLQILPVFFSNCLFLALYDSVILLKHVVLLLSRSKSTRGEWRRMLTSEGLRCVWKSFLLDAYKQVKLGEDAPNSSVVHVSSTEGGDNSGNGTQEKIAEGATCHLLDFASPERPLVVNFGSATUPPFTSQLPAFRKLVEEFSSVADFLLVYIDEAHPSDGWAIPGDSSLSFEVKKHQNQEDRCAAAQQLLERFSLPPQCRVVADRMDNNANIAYGVAFERVCIVQRQKIAYLGGKGPFSYNLQEVRHWLEKNFSKRUKKTRLAG .:.........:.........:.........:.........:.........:.........:.........:.........:.........86000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........87000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........88000 .........:.........:.....WARNING: Found the following zero-mass residues in protein entry sp|Q99611|SPS2_HUMAN : U MAEASATGACGEAMAAAEGSSGPAGLTLGRSFSNYRPFEPQALGLSPSWRLTGFSGMKGUGCKVPQEALLKLLAGLTRPDVRPPLGRGLVGGQEEASQEAGLPAGAGPSPTFPALGIGMDSCVIPLRHGGLSLVQTTDFFYPLVEDPYMMGRIACANVLSDLYAMGITECDNMLMLLSVSQSMSEEEREKVTPLMVKGFRDAAEEGGTAVTGGQTVVNPWIIIGGVATVVCQPNEFIMPDSAVVGDVLVLTKPLGTQVAVNAHQWLDNPERWNKVKMVVSREEVELAYQEAMFNMATLNRTAAGLMHTFNAHAATDITGFGILGHSQNLAKQQRNEVSFVIHNLPIIAKMAAVSKASGRFGLLQGTSAETSGGLLICLPREQAARFCSEIKSSKYGEGHQAWIVGIVEKGNRTARIIDKPRVIEVLPRGATAAVLAPDSSNASSEPSS ....:.........:.........:.........:.........:.........:.........:.........89000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........90000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........91000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........92000 .........:.........:...WARNING: Found the following zero-mass residues in protein entry sp|Q9NNW7-2|TRXR2_HUMAN : U MEDQRGAAAGQRDYDLLVVGGGSGGLACAKEAAQLGRKVAVVDYVEPSPQGTRWGLGGTCVNVGCIPKKLMHQAALLGGLIQDAPNYGWEVAQPVPHDWRKMAEAVQNHVKSLNWGHRVQLQDRKVKYFNIKASFVDEHTVCGVAKGGKEILLSADHIIIATGGRPRYPTHIEGALEYGITSDDIFWLKESPGKTLVVGASYVALECAGFLTGIGLDTTIMMRSIPLRGFDQQMSSMVIEHMASHGTRFLRGCAPSRVRRLPDGQLQVTWEDSTTGKEDTGTFDTVLWAIGRVPDTRSLNLEKAGVDTSPDTQKILVDSREATSVPHIYAIGDVVEGRPELTPIAIMAGRLLVQRLFGGSSDLMDYDNVPTTVFTPLEYGCVGLSEEEAVARHGQEHVEVYHAHYKPLEFTVAGRDASQCYVKMVCLREPPQLVLGLHFLGPNAGEVTQGFALGIKCGASYAQVMRTVGIHPTCSEEVVKLRISKRSGLDPTVTGCUG WARNING: Found the following zero-mass residues in protein entry sp|Q9NNW7|TRXR2_HUMAN : U MAAMAVALRGLGGRFRWRTQAVAGGVRGAARGAAAGQRDYDLLVVGGGSGGLACAKEAAQLGRKVAVVDYVEPSPQGTRWGLGGTCVNVGCIPKKLMHQAALLGGLIQDAPNYGWEVAQPVPHDWRKMAEAVQNHVKSLNWGHRVQLQDRKVKYFNIKASFVDEHTVCGVAKGGKEILLSADHIIIATGGRPRYPTHIEGALEYGITSDDIFWLKESPGKTLVVGASYVALECAGFLTGIGLDTTIMMRSIPLRGFDQQMSSMVIEHMASHGTRFLRGCAPSRVRRLPDGQLQVTWEDSTTGKEDTGTFDTVLWAIGRVPDTRSLNLEKAGVDTSPDTQKILVDSREATSVPHIYAIGDVVEGRPELTPIAIMAGRLLVQRLFGGSSDLMDYDNVPTTVFTPLEYGCVGLSEEEAVARHGQEHVEVYHAHYKPLEFTVAGRDASQCYVKMVCLREPPQLVLGLHFLGPNAGEVTQGFALGIKCGASYAQVMRTVGIHPTCSEEVVKLRISKRSGLDPTVTGCUG ......:.........:.........:.........:.........:.........:.........:.........93000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........94000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........95000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........96000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........97000 .........:.........:.........:.........:.........:.........:.........:.........:.........:......... Total: 97996 Computing degenerate peptides for 12684 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Computing probabilities for 15323 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15323 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15323 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15323 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15323 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 15323 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Calculating sensitivity...and error tables... INFO: mu=2.33361e-06, db_size=110649606 Computing MU for 15323 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% INFO[21:55:02] Done
Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe database --annotate C:\MS_Testrun_Raphael_126126\2022-08-12-decoys-contam-uniprot_SWP_homo_sapiens_26610entries05082022.fasta.fas --prefix rev INFO[21:55:02] Executing Database v4.5.1
INFO[21:55:02] Annotating the database
INFO[21:55:07] Done
Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopherv4.5.1-RC8\philosopher-v4.5.1-RC8.exe filter --sequential --picked --prot 0.01 --tag rev --pepxml C:\MS_Testrun_Raphael_126126 --protxml C:\MS_Testrun_Raphael_126126\combined.prot.xml --razor INFO[21:55:07] Executing Filter v4.5.1
INFO[21:55:07] Processing peptide identification files
INFO[21:55:07] Parsing C:\MS_Testrun_Raphael_126126\interact-Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1.pep.xml INFO[21:55:10] 1+ Charge profile decoy=0 target=0 INFO[21:55:10] 2+ Charge profile decoy=204 target=20183 INFO[21:55:10] 3+ Charge profile decoy=84 target=10476 INFO[21:55:10] 4+ Charge profile decoy=28 target=2357 INFO[21:55:10] 5+ Charge profile decoy=0 target=0 INFO[21:55:10] 6+ Charge profile decoy=0 target=0 INFO[21:55:10] Database search results ions=28695 peptides=25812 psms=33332 INFO[21:55:10] Converged to 0.95 % FDR with 33016 PSMs decoy=316 threshold=0.0504 total=33332 INFO[21:55:10] Converged to 1.00 % FDR with 25434 Peptides decoy=256 threshold=0.0873 total=25690 INFO[21:55:10] Converged to 1.00 % FDR with 28338 Ions decoy=286 threshold=0.0661 total=28624 INFO[21:55:12] Protein inference results decoy=359 target=6590 INFO[21:55:12] Converged to 1.01 % FDR with 4271 Proteins decoy=43 threshold=0.9256 total=4314 INFO[21:55:13] Applying sequential FDR estimation ions=27878 peptides=25010 psms=32496 INFO[21:55:13] Converged to 0.14 % FDR with 32449 PSMs decoy=47 threshold=0.0525 total=32496 INFO[21:55:13] Converged to 0.16 % FDR with 24969 Peptides decoy=41 threshold=0.5009 total=25010 INFO[21:55:13] Converged to 0.16 % FDR with 27832 Ions decoy=46 threshold=0.0525 total=27878 INFO[21:55:14] Post processing identifications
INFO[21:55:16] Assigning protein identifications to layers
INFO[21:55:17] Processing protein inference
INFO[21:55:38] Synchronizing PSMs and proteins
INFO[21:55:38] Total report numbers after FDR filtering, and post-processing ions=27827 peptides=24964 proteins=4271 psms=32442 INFO[21:55:38] Saving
INFO[21:55:39] Done
Process 'PhilosopherFilter' finished, exit code: 0 FreeQuant [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe freequant --ptw 0.4 --tol 10 --dir C:\MS_Testrun_Raphael_126126 INFO[21:55:39] Executing Label-free quantification v4.5.1
INFO[21:55:40] Indexing PSM information
INFO[21:55:40] Reading spectra and tracing peaks
INFO[21:55:40] Processing Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1 INFO[21:55:57] Assigning intensities to data layers
INFO[21:55:58] Done
Process 'FreeQuant' finished, exit code: 0 Quant (Isobaric) [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe labelquant --tol 20 --level 2 --plex 6 --annot C:\MS_Testrun_Raphael_126126\annotation.txt --brand tmt --dir C:\MS_Testrun_Raphael_126126 INFO[21:55:58] Executing Isobaric-label quantification v4.5.1 INFO[21:55:59] Calculating intensities and ion interference INFO[21:55:59] Processing Bert_220804_P9999_MR_RK_lysates_TMT4_1out60ul_R1 INFO[21:56:17] Filtering spectra for label quantification
INFO[21:56:17] Removing 0 PSMs from isobaric quantification INFO[21:56:17] Calculating normalized protein levels
INFO[21:56:17] Saving
INFO[21:56:18] Done
Process 'Quant (Isobaric)' finished, exit code: 0 PhilosopherReport [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe report INFO[21:56:19] Executing Report v4.5.1
INFO[21:56:19] Creating reports
INFO[21:56:22] Done
Process 'PhilosopherReport' finished, exit code: 0 WorkspaceClean [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\tools\philosopher_v4.5.1-RC8\philosopher-v4.5.1-RC8.exe workspace --clean --nocheck INFO[21:56:22] Executing Workspace v4.5.1
INFO[21:56:22] Removing workspace
INFO[21:56:22] Done
Process 'WorkspaceClean' finished, exit code: 0 TmtIntegrator [Work dir: C:\MS_Testrun_Raphael_126126] C:\Fragpipe\fragpipe\jre\bin\java.exe -Xmx46G -cp "C:\Fragpipe\fragpipe\tools\tmt-integrator-3.3.3.jar" TMTIntegrator C:\MS_Testrun_Raphael_126126\tmt-integrator-conf.yml C:\MS_Testrun_Raphael_126126\psm.tsv TMT-Integrator v3.3.3 UpdateColumns--- 0,01843 min. LoadPsms--- 0,00370 min. Take log and normalize--- 0,00133 min. PSM normalization--- 0,00000 min. outlierRemoval--- 0,00168 min. Collapse--- 0,00085 min. protNorm--- 0,00000 min. Report--- 0,00037 min. Finish!!! Process 'TmtIntegrator' finished, exit code: 0

Please cite: (Any searches) MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. Nat Methods 14:513 (2017) (Any searches) Fast deisotoping algorithm and its implementation in the MSFragger search engine. J. Proteome Res. 20:498 (2021) (Open search) Identification of modified peptides using localization-aware open search. Nat Commun. 11:4065 (2020) (Open search) Crystal-C: A Computational Tool for Refinement of Open Search Results. J. Proteome Res. 19.6:2511 (2020) (Open search) PTM-Shepherd: analysis and summarization of post-translational and chemical modifications from open search results. Mol Cell Proteomics 20:100018 (2020) (Glyco/labile search) Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco. Nat Methods 17:1125 (2020) (timsTOF PASEF) Fast quantitative analysis of timsTOF PASEF data with MSFragger and IonQuant. Mol Cell Proteomics 19:1575 (2020) (PSM validation with Percolator) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4:923 (2007) (Label-free quantification/SILAC) IonQuant Enables Accurate and Sensitive Label-Free Quantification With FDR-Controlled Match-Between-Runs. Mol Cell Proteomics 20:100077 (2021) (PeptideProphet/ProteinProphet/PTMProphet/Filtering) Philosopher: a versatile toolkit for shotgun proteomics data analysis. Nat Methods 17:869 (2020) (TMT-Integrator) Quantitative proteomic landscape of metaplastic breast carcinoma pathological subtypes and their relationship to triple-negative tumors. Nat Commun. 11:1723 (2020) (DIA-Umpire) DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics. Nat Methods 12:258 (2015) (DIA-NN) High sensitivity dia-PASEF proteomics with DIA-NN and FragPipe. bioRxiv doi:10.1101/2021.03.08.434385 (2021)

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fstein commented 2 years ago

Thansk a lot Felipe.