Error when running DIA_DIA-Umpire_SpecLib_Quant workflow

Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0. Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Please try using other options for alignment (e.g. ciRT if used other options) Process 'SpecLibGen' finished, exit code: 1 Process returned non-zero exit code, stopping

Is it human data? If not you need to use iRT alignment option as the message says

If hunan, we need to see the whole log

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From: yishai99 @.> Sent: Monday, February 6, 2023 3:08:26 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

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Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

— Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/993, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM64MQWJLCR7C5QMW5TLWWCWPVANCNFSM6AAAAAAUSLLM6A. You are receiving this because you are subscribed to this thread.Message ID: @.***>

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It is human serum.

See attached.

On 6 Feb 2023, at 15:31, Alexey Nesvizhskii @.***> wrote:

Is it human data? If not you need to use iRT alignment option as the message says

If hunan, we need to see the whole log

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From: yishai99 @.> Sent: Monday, February 6, 2023 3:08:26 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

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Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0. Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Please try using other options for alignment (e.g. ciRT if used other options) Process 'SpecLibGen' finished, exit code: 1 Process returned non-zero exit code, stopping

— Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/993, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM64MQWJLCR7C5QMW5TLWWCWPVANCNFSM6AAAAAAUSLLM6A. You are receiving this because you are subscribed to this thread.Message ID: @.***>

Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues — Reply to this email directly, view it on GitHub https://github.com/Nesvilab/FragPipe/issues/993#issuecomment-1419087186, or unsubscribe https://github.com/notifications/unsubscribe-auth/AE5PDY4YMCBK5WSQGSP7N2TWWD4KHANCNFSM6AAAAAAUSLLM6A. You are receiving this because you authored the thread.

System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK .NET Core Info: N/A

Version info: FragPipe version 19.1 MSFragger version 3.7 IonQuant version 1.8.10 Philosopher version 4.8.1

LCMS files: Experiment/Group:

D:\17915\FS1_MB_17915_2_29012023.raw DDA

17 commands to execute: CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx841G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.raw 63 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\6e27fc47-5e86-42e0-8695-aff8ae7739a7 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx841G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.raw MSFragger move pepxml C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pepXML D:\17915\Temp\FS1_MB_17915_2_29012023.pepXML MSFragger move pin C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pin D:\17915\Temp\FS1_MB_17915_2_29012023.pin MSBooster [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx841G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\msbooster-1.1.11.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" Features.MainClass --paramsList D:\17915\Temp\msbooster_params.txt Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt WorkspaceClean [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck


Execution order:

    Cmd: [START], Work dir: [D:\17915\Temp]
    Cmd: [CheckCentroid], Work dir: [D:\17915\Temp]
    Cmd: [WorkspaceCleanInit], Work dir: [D:\17915\Temp]
    Cmd: [MSFragger], Work dir: [D:\17915\Temp]
    Cmd: [MSBooster], Work dir: [D:\17915\Temp]
    Cmd: [Percolator], Work dir: [D:\17915\Temp]
    Cmd: [ProteinProphet], Work dir: [D:\17915\Temp]
    Cmd: [PhilosopherDbAnnotate], Work dir: [D:\17915\Temp]
    Cmd: [PhilosopherFilter], Work dir: [D:\17915\Temp]
    Cmd: [PhilosopherReport], Work dir: [D:\17915\Temp]
    Cmd: [SpecLibGen], Work dir: [D:\17915\Temp]
    Cmd: [WorkspaceClean], Work dir: [D:\17915\Temp]


>rev_sp|A0A024RBG1|NUD4B_HUMAN Diphosphoinositol polyphosphate phosphohydrolase NUDT4B OS=Homo sapiens OX=9606 GN=NUDT4B PE=3 SV=1
>rev_sp|O43908|NKG2F_HUMAN NKG2-F type II integral membrane protein OS=Homo sapiens OX=9606 GN=KLRC4 PE=2 SV=2
>rev_sp|P0DPK2|H3Y1_HUMAN Histone H3.Y OS=Homo sapiens OX=9606 GN=H3Y1 PE=1 SV=1
>rev_sp|P50336|PPOX_HUMAN Protoporphyrinogen oxidase OS=Homo sapiens OX=9606 GN=PPOX PE=1 SV=1
>rev_sp|Q14416|GRM2_HUMAN Metabotropic glutamate receptor 2 OS=Homo sapiens OX=9606 GN=GRM2 PE=1 SV=2
>rev_sp|Q6DKK2|TTC19_HUMAN Tetratricopeptide repeat protein 19, mitochondrial OS=Homo sapiens OX=9606 GN=TTC19 PE=1 SV=4
>rev_sp|Q8IX21|SLF2_HUMAN SMC5-SMC6 complex localization factor protein 2 OS=Homo sapiens OX=9606 GN=SLF2 PE=1 SV=2
>rev_sp|Q8WWH5|TRUB1_HUMAN Pseudouridylate synthase TRUB1 OS=Homo sapiens OX=9606 GN=TRUB1 PE=1 SV=1
>rev_sp|Q9BS26|ERP44_HUMAN Endoplasmic reticulum resident protein 44 OS=Homo sapiens OX=9606 GN=ERP44 PE=1 SV=1
>rev_sp|Q9NVH2|INT7_HUMAN Integrator complex subunit 7 OS=Homo sapiens OX=9606 GN=INTS7 PE=1 SV=1
>rev_sp|S4R3P1|HMN13_HUMAN Humanin-like 13 OS=Homo sapiens OX=9606 GN=MTRNR2L13 PE=3 SV=1
>sp|O43869|OR2T1_HUMAN Olfactory receptor 2T1 OS=Homo sapiens OX=9606 GN=OR2T1 PE=3 SV=3
>sp|P0DPF7|TVB63_HUMAN T cell receptor beta variable 6-3 OS=Homo sapiens OX=9606 GN=TRBV6-3 PE=3 SV=1
>sp|P50219|MNX1_HUMAN Motor neuron and pancreas homeobox protein 1 OS=Homo sapiens OX=9606 GN=MNX1 PE=1 SV=3
>sp|Q14390|GGTL2_HUMAN Glutathione hydrolase light chain 2 OS=Homo sapiens OX=9606 GN=GGTLC2 PE=1 SV=4
>sp|Q6DHV7|ADAL_HUMAN Adenosine deaminase-like protein OS=Homo sapiens OX=9606 GN=ADAL PE=1 SV=2
>sp|Q8IX05|CD302_HUMAN CD302 antigen OS=Homo sapiens OX=9606 GN=CD302 PE=1 SV=1
>sp|Q8WWF3|SSMM1_HUMAN Serine-rich single-pass membrane protein 1 OS=Homo sapiens OX=9606 GN=SSMEM1 PE=1 SV=1
>sp|Q9BRX5|PSF3_HUMAN DNA replication complex GINS protein PSF3 OS=Homo sapiens OX=9606 GN=GINS3 PE=1 SV=1
>sp|Q9NVE5|UBP40_HUMAN Ubiquitin carboxyl-terminal hydrolase 40 OS=Homo sapiens OX=9606 GN=USP40 PE=1 SV=3
>sp|Q9Y6Y8|S23IP_HUMAN SEC23-interacting protein OS=Homo sapiens OX=9606 GN=SEC23IP PE=1 SV=1

# FragPipe v19.1ui state cache

# Please edit the following path to point to the correct location.
# In Windows, please replace single '\' with '\\'
database.db-path=D\:\\Databases\\2023-01-25-decoys-contam-Human_SP_Jan2023.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=true
diann.run-specific-protein-q-value=false
diann.unrelated-runs=false
diann.use-predicted-spectra=true
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=true
fragpipe-config.bin-ionquant=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\IonQuant-1.8.10.jar
fragpipe-config.bin-msfragger=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\MSFragger-3.7\\MSFragger-3.7.jar
fragpipe-config.bin-philosopher=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\philosopher_v4.8.1_windows_amd64\\philosopher.exe
fragpipe-config.bin-python=C\:\\Users\\protolab\\Anaconda3\\python.exe

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=10
ionquant.medium=
ionquant.minfreq=0
ionquant.minions=1
ionquant.minisotopes=1
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=true
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=0/1/2
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=5
msfragger.min_sequence_matches=2
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.00
msfragger.misc.fragger.clear-mz-hi=0
msfragger.misc.fragger.clear-mz-lo=0
msfragger.misc.fragger.digest-mass-hi=5000
msfragger.misc.fragger.digest-mass-lo=500
msfragger.misc.fragger.enzyme-dropdown-1=stricttrypsin
msfragger.misc.fragger.enzyme-dropdown-2=null
msfragger.misc.fragger.precursor-charge-hi=4
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=stricttrypsin
msfragger.search_enzyme_name_2=null
msfragger.search_enzyme_nocut_1=
msfragger.search_enzyme_nocut_2=
msfragger.search_enzyme_sense_1=C
msfragger.search_enzyme_sense_2=C
msfragger.table.fix-mods=0.0,C-Term Peptide,true,-1; 0.0,N-Term Peptide,true,-1; 0.0,C-Term Protein,true,-1; 0.0,N-Term Protein,true,-1; 0.0,G (glycine),true,-1; 0.0,A (alanine),true,-1; 0.0,S (serine),true,-1; 0.0,P (proline),true,-1; 0.0,V (valine),true,-1; 0.0,T (threonine),true,-1; 57.02146,C (cysteine),true,-1; 0.0,L (leucine),true,-1; 0.0,I (isoleucine),true,-1; 0.0,N (asparagine),true,-1; 0.0,D (aspartic acid),true,-1; 0.0,Q (glutamine),true,-1; 0.0,K (lysine),true,-1; 0.0,E (glutamic acid),true,-1; 0.0,M (methionine),true,-1; 0.0,H (histidine),true,-1; 0.0,F (phenylalanine),true,-1; 0.0,R (arginine),true,-1; 0.0,Y (tyrosine),true,-1; 0.0,W (tryptophan),true,-1; 0.0,B ,true,-1; 0.0,J,true,-1; 0.0,O,true,-1; 0.0,U,true,-1; 0.0,X,true,-1; 0.0,Z,true,-1
msfragger.table.var-mods=15.9949,M,true,3; 42.0106,[^,true,1; 79.96633,STY,false,3; -17.0265,nQnC,true,1; -18.0106,nE,true,1; 0.0,site_06,false,1; 0.0,site_07,false,1; 0.0,site_08,false,1; 0.0,site_09,false,1; 0.0,site_10,false,1; 0.0,site_11,false,1; 0.0,site_12,false,1; 0.0,site_13,false,1; 0.0,site_14,false,1; 0.0,site_15,false,1; 0.0,site_16,false,1
msfragger.track_zero_topN=0
msfragger.use_all_mods_in_first_search=false
msfragger.use_topN_peaks=300
msfragger.write_calibrated_mzml=false
msfragger.write_uncalibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
opair.activation1=HCD
opair.activation2=ETD
opair.glyco_db=
opair.max_glycans=4
opair.max_isotope_error=2
opair.min_isotope_error=0
opair.ms1_tol=20
opair.ms2_tol=20
opair.reverse_scan_order=false
opair.run-opair=false
opair.single_scan_type=false
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=false
percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc

percolator.keep-tsv-files=false
percolator.min-prob=0.7
percolator.run-percolator=true
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--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=true
phi-report.remove-contaminants=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.cap_y_ions=
ptmshepherd.decoy_type=1
ptmshepherd.diag_ions=
ptmshepherd.diagextract_mode=false
ptmshepherd.diagmine_diagMinFoldChange=3.0
ptmshepherd.diagmine_diagMinSpecDiff=25
ptmshepherd.diagmine_fragMinFoldChange=3.0
ptmshepherd.diagmine_fragMinPropensity=12.5
ptmshepherd.diagmine_fragMinSpecDiff=25
ptmshepherd.diagmine_minIonsPerSpec=2
ptmshepherd.diagmine_minPeps=25
ptmshepherd.diagmine_mode=false
ptmshepherd.diagmine_pepMinFoldChange=3.0
ptmshepherd.diagmine_pepMinSpecDiff=25
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=2,0.5,0.1
ptmshepherd.prob_dhexY=2,0.5
ptmshepherd.prob_neuacOx=2,0.05,0.2
ptmshepherd.prob_neugcOx=2,0.05,0.2
ptmshepherd.prob_phosphoOx=2,0.05,0.2
ptmshepherd.prob_regY=5,0.5
ptmshepherd.prob_sulfoOx=2,0.05,0.2
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
saintexpress.fragpipe.cmd-opts=
saintexpress.max-replicates=10
saintexpress.run-saint-express=false
saintexpress.virtual-controls=100
speclibgen.easypqp.extras.max_delta_ppm=15
speclibgen.easypqp.extras.max_delta_unimod=0.02
speclibgen.easypqp.extras.rt_lowess_fraction=0
speclibgen.easypqp.fragment.a=false
speclibgen.easypqp.fragment.b=true
speclibgen.easypqp.fragment.c=false
speclibgen.easypqp.fragment.x=false
speclibgen.easypqp.fragment.y=true
speclibgen.easypqp.fragment.z=false
speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM

speclibgen.easypqp.neutral_loss=false
speclibgen.easypqp.rt-cal=noiRT
speclibgen.easypqp.select-file.text=
speclibgen.easypqp.select-im-file.text=
speclibgen.keep-intermediate-files=false
speclibgen.run-speclibgen=true
tab-run.delete_calibrated_mzml=false
tab-run.delete_temp_files=false
tmtintegrator.add_Ref=-1
tmtintegrator.aggregation_method=0
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=TMT-6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.glyco_qval=-1
tmtintegrator.groupby=0
tmtintegrator.log2transformed=true
tmtintegrator.max_pep_prob_thres=0
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=Bridge
tmtintegrator.run-tmtintegrator=false
tmtintegrator.tolerance=20
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=0
tmtintegrator.unique_pep=false
tmtintegrator.use_glycan_composition=false
workdir=D\:\\17915\\Temp
workflow.input.data-type.im-ms=false
workflow.input.data-type.regular-ms=true
workflow.ram=0
workflow.threads=63

CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx841G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.raw 63 Done in 0 ms. Process 'CheckCentroid' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck INFO[15:07:46] Executing Workspace v4.8.1
INFO[15:07:46] Removing workspace
INFO[15:07:46] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\6e27fc47-5e86-42e0-8695-aff8ae7739a7 INFO[15:07:47] Executing Workspace v4.8.1
INFO[15:07:47] Creating workspace
INFO[15:07:47] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx841G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.raw MSFragger version MSFragger-3.7 Batmass-IO version 1.28.9 timsdata library version timsdata-2-21-0-4 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. timdTOF .d reading tool. Copyright (c) 2022 by Bruker Daltonics GmbH & Co. KG. All rights reserved. System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK JVM started with 841 GB memory Checking database... Checking spectral files... D:\17915\FS1_MB_17915_2_29012023.raw: Scans = 84704 ***FIRST SEARCH**** Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 5 minimum_ratio = 0.0 intensity_transform = 0 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.10 Da. 344926862 fragments to be searched in 1 slices (5.14 GB total) Operating on slice 1 of 1: Fragment index slice generated in 4.59 s

FS1_MB_17915_2_29012023.raw 74.8 s | deisotoping 3.9 s [progress: 84420/84420 (100%) - 16179 spectra/s] 5.2s | postprocessing 0.6 s ***FIRST SEARCH DONE IN 2.019 MIN**

***MASS CALIBRATION AND PARAMETER OPTIMIZATION* -----	---------------	---------------	---------------	---------------
Run	Median MAD	Median MAD	Median MAD	Median MAD
001	Not enough data to perform mass calibration. Using the uncalibrated data.
-----	---------------	---------------	---------------	---------------

Finding the optimal parameters: -------|-------|-------|-------|-------|-------|-------|-------|-------

MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50

-------|-------|-------|-------|-------|-------|-------|-------|-------

Count	5	29	34	42	39	skip rest

-------	-------	-------	-------	-------	-------	------- Peaks
Count	42	41	skip rest
-------	-------	-------	-------	-------	-------	-------
-------	-------
Int.	1
-------	-------
Count	45
-------	-------
-------	-------
Rm P.	0
-------	-------
Count	48
-------	-------

New fragment_mass_tolerance = 15 PPM New use_topN_peaks = 300 New minimum_ratio = 0.000000 New intensity_transform = 1 New remove_precursor_peak = 0 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 7.541 MIN*****

****MAIN SEARCH**** Checking database... Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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 = 15.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = false write_calibrated_mzml = 0 write_uncalibrated_mgf = false isotope_error = 0/1/2 mass_offsets = 0.0 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 5 minimum_ratio = 0.0 intensity_transform = 1 activation_types = all remove_precursor_peak = 0 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 variable_mod_05 = -18.0106 nE 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.08 Da. 368492928 fragments to be searched in 1 slices (5.49 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.56 s

FS1_MB_17915_2_29012023.raw 45.8 s | deisotoping 2.6 s [progress: 84420/84420 (100%) - 36451 spectra/s] 2.3s | remapping alternative proteins and postprocessing 6.8 s MAIN SEARCH DONE IN 1.099 MIN

***TOTAL TIME 10.660 MIN**** Process 'MSFragger' finished, exit code: 0 MSFragger move pepxml C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pepXML D:\17915\Temp\FS1_MB_17915_2_29012023.pepXML Process 'MSFragger move pepxml' finished, exit code: 0 MSFragger move pin C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pin D:\17915\Temp\FS1_MB_17915_2_29012023.pin Process 'MSFragger move pin' finished, exit code: 0 MSBooster [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx841G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\msbooster-1.1.11.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" Features.MainClass --paramsList D:\17915\Temp\msbooster_params.txt MSBooster v1.1.11 Using 63 threads Generating input file for DIA-NN 5094 unique peptides from 19426 PSMs Writing DIA-NN input file Diann input file generation took 1210 milliseconds Input file at D:\17915\Temp\spectraRT.tsv 5094 unique peptides from 19426 PSMs createFull input file generation took 262 milliseconds Input file at D:\17915\Temp\spectraRT_full.tsv Generating DIA-NN predictions C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\diann\1.8.2_beta_8\win\DiaNN.exe --lib D:\17915\Temp\spectraRT.tsv --predict --threads 63 --strip-unknown-mods --mod TMT,229.1629 --predict-n-frag 100 DIA-NN 1.8.2 beta 8 (Data-Independent Acquisition by Neural Networks) Compiled on Sep 15 2022 18:28:57 Current date and time: Mon Feb 6 15:18:32 2023 CPU: AuthenticAMD AMD EPYC 7552 48-Core Processor SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2 SSE4a Logical CPU cores: 64 Predicted spectra will be saved in a binary format Thread number set to 63 DIA-NN will use deep learning to predict spectra/RTs/IMs even for peptides carrying modifications which are not recognised by the deep learning predictor. In this scenario, if also generating a spectral library from the DIA data or using the MBR mode, it might or might not be better (depends on the data) to also use the --out-measured-rt option - it's recommended to test it with and without this option Modification TMT with mass delta 229.163 added to the list of recognised modifications for spectral library-based search Deep learning predictor will predict 100 fragments Cannot find a UniMod modification match for TMT: 73.0618 minimal mass discrepancy; using the original modificaiton name

0 files will be processed [0:00] Loading spectral library D:\17915\Temp\spectraRT.tsv [0:00] Finding proteotypic peptides (assuming that the list of UniProt ids provided for each peptide is complete) [0:00] Spectral library loaded: 0 protein isoforms, 0 protein groups and 5094 precursors in 5094 elution groups. [0:00] Encoding peptides for spectra and RTs prediction [0:00] Predicting spectra and IMs [0:08] Predicting RTs [0:10] Decoding predicted spectra and IMs [0:11] Decoding RTs [0:11] Saving the list of predictions to D:\17915\Temp\spectraRT.predicted.bin Finished Done generating DIA-NN predictions Model running took 12395 milliseconds Generating edited pin with following features: [unweightedSpectralEntropy, deltaRTLOESS] Loading predicted spectra Processing FS1_MB_17915_2_29012023_uncalibrated.mzML RT regression using 5 PSMs Edited pin file at D:\17915\Temp\FS1_MB_17915_2_29012023_edited.pin Done in 14544 ms Process 'MSBooster' finished, exit code: 0 Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator version 3.05.0, Build Date May 18 2020 02:35:01 Copyright (c) 2006-9 University of Washington. All rights reserved. Written by Lukas Käll @.***) in the Department of Genome Sciences at the University of Washington. Issued command: C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Started Mon Feb 6 15:18:58 2023 Hyperparameters: selectionFdr=0.01, Cpos=0, Cneg=0, maxNiter=10

Reading tab-delimited input from datafile FS1_MB_17915_2_29012023_edited.pin Features: retentiontime rank abs_ppm isotope_errors log10_evalue hyperscore delta_hyperscore matched_ion_num complementary_ions ion_series weighted_average_abs_fragment_ppm peptide_length ntt nmc charge_1 charge_2 charge_3 charge_4 charge_5 charge_6 charge_7_or_more -17.0265nQnC -18.0106nE 15.9949M unweighted_spectral_entropy delta_RT_loess Found 19426 PSMs Concatenated search input detected and --post-processing-tdc flag set. Applying target-decoy competition on Percolator scores. Train/test set contains 9602 positives and 9824 negatives, size ratio=0.977402 and pi0=1 Selecting Cpos by cross-validation. Selecting Cneg by cross-validation. Split 1: Selected feature 5 as initial direction. Could separate 25 training set positives with q<0.01 in that direction. Split 2: Selected feature 25 as initial direction. Could separate 29 training set positives with q<0.01 in that direction. Split 3: Selected feature 25 as initial direction. Could separate 31 training set positives with q<0.01 in that direction. Found 0 test set positives with q<0.01 in initial direction Reading in data and feature calculation took 0.973 cpu seconds or 1 seconds wall clock time. ---Training with Cpos selected by cross validation, Cneg selected by cross validation, initial_fdr=0.01, fdr=0.01 Iteration 1: Estimated 0 PSMs with q<0.01 Iteration 2: Estimated 63 PSMs with q<0.01 Iteration 3: Estimated 75 PSMs with q<0.01 Iteration 4: Estimated 134 PSMs with q<0.01 Iteration 5: Estimated 162 PSMs with q<0.01 Iteration 6: Estimated 170 PSMs with q<0.01 Iteration 7: Estimated 173 PSMs with q<0.01 Iteration 8: Estimated 178 PSMs with q<0.01 Iteration 9: Estimated 184 PSMs with q<0.01 Iteration 10: Estimated 238 PSMs with q<0.01 Learned normalized SVM weights for the 3 cross-validation splits: Split1 Split2 Split3 FeatureName 0.5675 1.4778 0.6368 retentiontime 0.0000 0.0000 0.0000 rank 1.4141 2.5560 -0.2722 abs_ppm 1.7811 -1.1856 0.1159 isotope_errors -2.4619 -1.7395 -0.5172 log10_evalue 0.7997 1.2280 0.9256 hyperscore -0.3012 0.2287 -0.1290 delta_hyperscore -2.5766 -2.0025 -0.8079 matched_ion_num 0.9646 -0.4274 -0.1799 complementary_ions 0.2328 0.4208 0.0568 ion_series -0.6094 -0.9183 -0.1758 weighted_average_abs_fragment_ppm -0.3891 0.3562 -0.5756 peptide_length 0.0000 0.0000 0.0000 ntt -0.1550 -5.3234 -0.0270 nmc 0.0000 0.0000 0.0000 charge_1 0.0000 0.0000 0.0000 charge_2 0.0000 0.0000 0.0000 charge_3 0.0000 0.0000 0.0000 charge_4 0.0000 0.0000 0.0000 charge_5 0.0000 0.0000 0.0000 charge_6 0.0000 0.0000 0.0000 charge_7_or_more -0.0390 -0.3477 -0.2676 -17.0265nQnC 0.0622 0.2002 -0.0384 -18.0106nE 0.4144 1.4449 -0.9616 15.9949M 1.4463 1.6179 1.3509 unweighted_spectral_entropy 0.2516 0.0542 -0.0024 delta_RT_loess -9.6143 -16.7652 -5.6070 m0 Found 0 test set PSMs with q<0.01. No targets found with q<0.01 Resetting score vector, using default vector. Use --override flag to prevent this. Split 1: Selected feature 5 as initial direction. Could separate 25 training set positives with q<0.01 in that direction. Split 2: Selected feature 25 as initial direction. Could separate 29 training set positives with q<0.01 in that direction. Split 3: Selected feature 25 as initial direction. Could separate 31 training set positives with q<0.01 in that direction. Selected best-scoring PSM per scan+expMass (target-decoy competition): 9602 target PSMs and 9824 decoy PSMs. Calculating q values. Final list yields 0 target PSMs with q<0.01. Calculating posterior error probabilities (PEPs). Processing took 7.857 cpu seconds or 8 seconds wall clock time. Process 'Percolator' finished, exit code: 0 Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Process 'Percolator: Convert to pepxml' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt INFO[15:19:14] Executing ProteinProphet v4.8.1
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 D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml... ...read in 0 1+, 0 2+, 31 3+, 0 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.5

Initializing 4 peptide weights: 0%...50%...100% Calculating protein lengths and molecular weights from database D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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 .........:.........:.........:.........:.........:.........:.........:.........:....... Total: 40874 Computing degenerate peptides for 4 proteins: 0%...50%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Calculating sensitivity...and error tables... INFO: mu=5.55363e-09, db_size=45578978 Computing MU for 4 proteins: 0%...50%...100% INFO[15:19:17] Done
Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev INFO[15:19:17] Executing Database v4.8.1
INFO[15:19:17] Annotating the database
INFO[15:19:23] Done
Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor INFO[15:19:23] Executing Filter v4.8.1
INFO[15:19:23] Processing peptide identification files
INFO[15:19:23] Parsing D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml INFO[15:19:23] 1+ Charge profile decoy=0 target=0 INFO[15:19:23] 2+ Charge profile decoy=0 target=0 INFO[15:19:23] 3+ Charge profile decoy=0 target=31 INFO[15:19:23] 4+ Charge profile decoy=0 target=0 INFO[15:19:23] 5+ Charge profile decoy=0 target=0 INFO[15:19:23] 6+ Charge profile decoy=0 target=0 INFO[15:19:23] Database search results ions=4 peptides=4 psms=31 INFO[15:19:23] Converged to 0.00 % FDR with 31 PSMs decoy=0 threshold=0.700492 total=31 INFO[15:19:23] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.854989 total=4 INFO[15:19:23] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.854989 total=4 INFO[15:19:23] Protein inference results decoy=0 target=4 INFO[15:19:23] Converged to 0.00 % FDR with 4 Proteins decoy=0 threshold=0.8541 total=4 INFO[15:19:23] 2D FDR estimation: Protein mirror image decoy=4 target=4 INFO[15:19:23] Second filtering results ions=4 peptides=4 psms=31 INFO[15:19:23] Converged to 0.00 % FDR with 31 PSMs decoy=0 threshold=0.700492 total=31 INFO[15:19:23] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.854989 total=4 INFO[15:19:23] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.854989 total=4 INFO[15:19:23] Post processing identifications
INFO[15:19:24] Assigning protein identifications to layers
INFO[15:19:25] Processing protein inference
INFO[15:19:26] Synchronizing PSMs and proteins
INFO[15:19:26] Final report numbers after FDR filtering, and post-processing ions=4 peptides=4 proteins=4 psms=31 INFO[15:19:26] Saving
INFO[15:19:26] Done
Process 'PhilosopherFilter' finished, exit code: 0 PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report INFO[15:19:26] Executing Report v4.8.1
INFO[15:19:26] Creating reports
INFO[15:19:26] Done
Process 'PhilosopherReport' finished, exit code: 0 SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt File list provided Using (un)calibrated.mzML files. Spectral library building Commands to execute: 'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' convert --max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types '['"'"'b'"'"','"'"'y'"'"',]' --enable_unannotated --pepxml 'D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml' --spectra 'D:\17915\FS1_MB_17915_2_29012023_uncalibrated.mzML' --exclude-range -1.5,3.5 --psms FS1_MB_17915_2_29012023.psmpkl --peaks FS1_MB_17915_2_29012023.peakpkl 'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' library --psmtsv 'D:\17915\Temp\psm.tsv' --peptidetsv 'D:\17915\Temp\peptide.tsv' --out easypqp_lib_openswath.tsv --rt_lowess_fraction 0.0 'D:\17915\Temp\filelist_easypqp_library.txt'

easypqp, version 0.1.35
Executing ['C:\\Users\\protolab\\Anaconda3\\Scripts\\easypqp.exe', 'convert', '--max_delta_unimod', '0.02', '--max_delta_ppm', '15.0', '--fragment_types', "['b','y',]", '--enable_unannotated', '--pepxml', 'D:\\17915\\Temp\\interact-FS1_MB_17915_2_29012023.pep.xml', '--spectra', 'D:\\17915\\FS1_MB_17915_2_29012023_uncalibrated.mzML', '--exclude-range', '-1.5,3.5', '--psms', 'FS1_MB_17915_2_29012023.psmpkl', '--peaks', 'FS1_MB_17915_2_29012023.peakpkl']
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
    return self._engine.get_loc(casted_key)
  File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
  File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
    return _run_code(code, main_globals, None,
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

    exec(code, run_globals)
  File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
    return self.main(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
    rv = self.invoke(ctx)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
    rv.append(sub_ctx.command.invoke(sub_ctx))
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
    return __callback(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
    generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
    pepida = pepida.loc[np.isfinite(pepida['irt'])]
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
    indexer = self.columns.get_loc(key)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
    raise KeyError(key) from err
KeyError: 'irt'
                 base_name  modified_peptide
0  FS1_MB_17915_2_29012023                 4
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 4.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Not enough peptides could be found for alignment.
Using ciRT for alignment
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
    return self._engine.get_loc(casted_key)
  File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
  File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
    return _run_code(code, main_globals, None,
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

    exec(code, run_globals)
  File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
    return self.main(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
    rv = self.invoke(ctx)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
    rv.append(sub_ctx.command.invoke(sub_ctx))
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
    return __callback(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
    generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
    pepida = pepida.loc[np.isfinite(pepida['irt'])]
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
    indexer = self.columns.get_loc(key)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
    raise KeyError(key) from err
KeyError: 'irt'
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Library not generated, not enough peptides could be found for alignment.

Library not generated, not enough peptides could be found for alignment.

Please try using other options for alignment (e.g. ciRT if used other options)
Process 'SpecLibGen' finished, exit code: 1
Process returned non-zero exit code, stopping

Cancelling 2 remaining tasks

You did something wrong. I think you wanted to use DIA-Umpire workflow but did not click LOAD Or maybe you did, but did not annotate the file as DIA type (it is DDA by default).

Also, for full DIA workflow (that includes DIA-NN for quant) I suggest you convert to mzML

Alexey

From: Levin @.> Sent: Monday, February 6, 2023 8:40 AM To: Nesvilab/FragPipe @.> Cc: Nesvilab/FragPipe @.>; Author @.>; Nesvizhskii, Alexey @.>; Fengchao Yu @.> Subject: Re: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

It is human serum.

See attached.

On 6 Feb 2023, at 15:31, Alexey Nesvizhskii @.**@.>> wrote:

Is it human data? If not you need to use iRT alignment option as the message says

If hunan, we need to see the whole log

Get Outlook for iOShttps://aka.ms/o0ukef

From: yishai99 @.<mailto:@.>> Sent: Monday, February 6, 2023 3:08:26 AM To: Nesvilab/FragPipe @.<mailto:@.>> Cc: Subscribed @.<mailto:@.>> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

— Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/993#issuecomment-1419087186, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AE5PDY4YMCBK5WSQGSP7N2TWWD4KHANCNFSM6AAAAAAUSLLM6A. You are receiving this because you authored the thread.Message ID: @.**@.>>

Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

There is some issue with Github system, so I am responding again in case you did not receive it

You did something wrong. I think you wanted to use DIA-Umpire workflow but did not click LOAD Or maybe you did, but did not annotate the file as DIA type (it is DDA by default).

Also, for full DIA workflow (that includes DIA-NN for quant) I suggest you convert to mzML

Alexey

From: yishai99 @.> Sent: Monday, February 6, 2023 3:08 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues

I tried again, this time making sure to Load the right workflow. Still get an error (attached).

On 6 Feb 2023, at 17:58, Alexey Nesvizhskii @.***> wrote:

There is some issue with Github system, so I am responding again in case you did not receive it

You did something wrong. I think you wanted to use DIA-Umpire workflow but did not click LOAD Or maybe you did, but did not annotate the file as DIA type (it is DDA by default).

Also, for full DIA workflow (that includes DIA-NN for quant) I suggest you convert to mzML

Alexey

From: yishai99 @.> Sent: Monday, February 6, 2023 3:08 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0. Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Please try using other options for alignment (e.g. ciRT if used other options) Process 'SpecLibGen' finished, exit code: 1 Process returned non-zero exit code, stopping

— Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/993, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM64MQWJLCR7C5QMW5TLWWCWPVANCNFSM6AAAAAAUSLLM6A. You are receiving this because you are subscribed to this thread.Message ID: @.**@.>>

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System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK .NET Core Info: N/A

Version info: FragPipe version 19.1 MSFragger version 3.7 IonQuant version 1.8.10 Philosopher version 4.8.1

LCMS files: Experiment/Group:

D:\17915\FS1_MB_17915_2_29012023.mzML DDA

17 commands to execute: CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.mzML 63 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\e92b1203-0a35-4118-baee-77b879b2cf05 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx844G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.mzML MSFragger move pepxml C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pepXML D:\17915\Temp\FS1_MB_17915_2_29012023.pepXML MSFragger move pin C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pin D:\17915\Temp\FS1_MB_17915_2_29012023.pin MSBooster [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\msbooster-1.1.11.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" Features.MainClass --paramsList D:\17915\Temp\msbooster_params.txt Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt WorkspaceClean [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck


Execution order:

    Cmd: [START], Work dir: [D:\17915\Temp]
    Cmd: [CheckCentroid], Work dir: [D:\17915\Temp]
    Cmd: [WorkspaceCleanInit], Work dir: [D:\17915\Temp]
    Cmd: [MSFragger], Work dir: [D:\17915\Temp]
    Cmd: [MSBooster], Work dir: [D:\17915\Temp]
    Cmd: [Percolator], Work dir: [D:\17915\Temp]
    Cmd: [ProteinProphet], Work dir: [D:\17915\Temp]
    Cmd: [PhilosopherDbAnnotate], Work dir: [D:\17915\Temp]
    Cmd: [PhilosopherFilter], Work dir: [D:\17915\Temp]
    Cmd: [PhilosopherReport], Work dir: [D:\17915\Temp]
    Cmd: [SpecLibGen], Work dir: [D:\17915\Temp]
    Cmd: [WorkspaceClean], Work dir: [D:\17915\Temp]


>rev_sp|A0A024RBG1|NUD4B_HUMAN Diphosphoinositol polyphosphate phosphohydrolase NUDT4B OS=Homo sapiens OX=9606 GN=NUDT4B PE=3 SV=1
>rev_sp|O43908|NKG2F_HUMAN NKG2-F type II integral membrane protein OS=Homo sapiens OX=9606 GN=KLRC4 PE=2 SV=2
>rev_sp|P0DPK2|H3Y1_HUMAN Histone H3.Y OS=Homo sapiens OX=9606 GN=H3Y1 PE=1 SV=1
>rev_sp|P50336|PPOX_HUMAN Protoporphyrinogen oxidase OS=Homo sapiens OX=9606 GN=PPOX PE=1 SV=1
>rev_sp|Q14416|GRM2_HUMAN Metabotropic glutamate receptor 2 OS=Homo sapiens OX=9606 GN=GRM2 PE=1 SV=2
>rev_sp|Q6DKK2|TTC19_HUMAN Tetratricopeptide repeat protein 19, mitochondrial OS=Homo sapiens OX=9606 GN=TTC19 PE=1 SV=4
>rev_sp|Q8IX21|SLF2_HUMAN SMC5-SMC6 complex localization factor protein 2 OS=Homo sapiens OX=9606 GN=SLF2 PE=1 SV=2
>rev_sp|Q8WWH5|TRUB1_HUMAN Pseudouridylate synthase TRUB1 OS=Homo sapiens OX=9606 GN=TRUB1 PE=1 SV=1
>rev_sp|Q9BS26|ERP44_HUMAN Endoplasmic reticulum resident protein 44 OS=Homo sapiens OX=9606 GN=ERP44 PE=1 SV=1
>rev_sp|Q9NVH2|INT7_HUMAN Integrator complex subunit 7 OS=Homo sapiens OX=9606 GN=INTS7 PE=1 SV=1
>rev_sp|S4R3P1|HMN13_HUMAN Humanin-like 13 OS=Homo sapiens OX=9606 GN=MTRNR2L13 PE=3 SV=1
>sp|O43869|OR2T1_HUMAN Olfactory receptor 2T1 OS=Homo sapiens OX=9606 GN=OR2T1 PE=3 SV=3
>sp|P0DPF7|TVB63_HUMAN T cell receptor beta variable 6-3 OS=Homo sapiens OX=9606 GN=TRBV6-3 PE=3 SV=1
>sp|P50219|MNX1_HUMAN Motor neuron and pancreas homeobox protein 1 OS=Homo sapiens OX=9606 GN=MNX1 PE=1 SV=3
>sp|Q14390|GGTL2_HUMAN Glutathione hydrolase light chain 2 OS=Homo sapiens OX=9606 GN=GGTLC2 PE=1 SV=4
>sp|Q6DHV7|ADAL_HUMAN Adenosine deaminase-like protein OS=Homo sapiens OX=9606 GN=ADAL PE=1 SV=2
>sp|Q8IX05|CD302_HUMAN CD302 antigen OS=Homo sapiens OX=9606 GN=CD302 PE=1 SV=1
>sp|Q8WWF3|SSMM1_HUMAN Serine-rich single-pass membrane protein 1 OS=Homo sapiens OX=9606 GN=SSMEM1 PE=1 SV=1
>sp|Q9BRX5|PSF3_HUMAN DNA replication complex GINS protein PSF3 OS=Homo sapiens OX=9606 GN=GINS3 PE=1 SV=1
>sp|Q9NVE5|UBP40_HUMAN Ubiquitin carboxyl-terminal hydrolase 40 OS=Homo sapiens OX=9606 GN=USP40 PE=1 SV=3
>sp|Q9Y6Y8|S23IP_HUMAN SEC23-interacting protein OS=Homo sapiens OX=9606 GN=SEC23IP PE=1 SV=1

# FragPipe v19.1ui state cache

# Please edit the following path to point to the correct location.
# In Windows, please replace single '\' with '\\'
database.db-path=D\:\\Databases\\2023-01-25-decoys-contam-Human_SP_Jan2023.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=true
diann.run-specific-protein-q-value=false
diann.unrelated-runs=false
diann.use-predicted-spectra=true
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=true
fragpipe-config.bin-ionquant=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\IonQuant-1.8.10.jar
fragpipe-config.bin-msfragger=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\MSFragger-3.7\\MSFragger-3.7.jar
fragpipe-config.bin-philosopher=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\philosopher_v4.8.1_windows_amd64\\philosopher.exe
fragpipe-config.bin-python=C\:\\Users\\protolab\\Anaconda3\\python.exe

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=10
ionquant.medium=
ionquant.minfreq=0
ionquant.minions=1
ionquant.minisotopes=1
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=true
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=0/1/2
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=5
msfragger.min_sequence_matches=2
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.00
msfragger.misc.fragger.clear-mz-hi=0
msfragger.misc.fragger.clear-mz-lo=0
msfragger.misc.fragger.digest-mass-hi=5000
msfragger.misc.fragger.digest-mass-lo=500
msfragger.misc.fragger.enzyme-dropdown-1=stricttrypsin
msfragger.misc.fragger.enzyme-dropdown-2=null
msfragger.misc.fragger.precursor-charge-hi=4
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=stricttrypsin
msfragger.search_enzyme_name_2=null
msfragger.search_enzyme_nocut_1=
msfragger.search_enzyme_nocut_2=
msfragger.search_enzyme_sense_1=C
msfragger.search_enzyme_sense_2=C
msfragger.table.fix-mods=0.0,C-Term Peptide,true,-1; 0.0,N-Term Peptide,true,-1; 0.0,C-Term Protein,true,-1; 0.0,N-Term Protein,true,-1; 0.0,G (glycine),true,-1; 0.0,A (alanine),true,-1; 0.0,S (serine),true,-1; 0.0,P (proline),true,-1; 0.0,V (valine),true,-1; 0.0,T (threonine),true,-1; 57.02146,C (cysteine),true,-1; 0.0,L (leucine),true,-1; 0.0,I (isoleucine),true,-1; 0.0,N (asparagine),true,-1; 0.0,D (aspartic acid),true,-1; 0.0,Q (glutamine),true,-1; 0.0,K (lysine),true,-1; 0.0,E (glutamic acid),true,-1; 0.0,M (methionine),true,-1; 0.0,H (histidine),true,-1; 0.0,F (phenylalanine),true,-1; 0.0,R (arginine),true,-1; 0.0,Y (tyrosine),true,-1; 0.0,W (tryptophan),true,-1; 0.0,B ,true,-1; 0.0,J,true,-1; 0.0,O,true,-1; 0.0,U,true,-1; 0.0,X,true,-1; 0.0,Z,true,-1
msfragger.table.var-mods=15.9949,M,true,3; 42.0106,[^,true,1; 79.96633,STY,false,3; -17.0265,nQnC,true,1; -18.0106,nE,true,1; 0.0,site_06,false,1; 0.0,site_07,false,1; 0.0,site_08,false,1; 0.0,site_09,false,1; 0.0,site_10,false,1; 0.0,site_11,false,1; 0.0,site_12,false,1; 0.0,site_13,false,1; 0.0,site_14,false,1; 0.0,site_15,false,1; 0.0,site_16,false,1
msfragger.track_zero_topN=0
msfragger.use_all_mods_in_first_search=false
msfragger.use_topN_peaks=300
msfragger.write_calibrated_mzml=false
msfragger.write_uncalibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
opair.activation1=HCD
opair.activation2=ETD
opair.glyco_db=
opair.max_glycans=4
opair.max_isotope_error=2
opair.min_isotope_error=0
opair.ms1_tol=20
opair.ms2_tol=20
opair.reverse_scan_order=false
opair.run-opair=false
opair.single_scan_type=false
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=false
percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc

percolator.keep-tsv-files=false
percolator.min-prob=0.7
percolator.run-percolator=true
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--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=true
phi-report.remove-contaminants=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.cap_y_ions=
ptmshepherd.decoy_type=1
ptmshepherd.diag_ions=
ptmshepherd.diagextract_mode=false
ptmshepherd.diagmine_diagMinFoldChange=3.0
ptmshepherd.diagmine_diagMinSpecDiff=25
ptmshepherd.diagmine_fragMinFoldChange=3.0
ptmshepherd.diagmine_fragMinPropensity=12.5
ptmshepherd.diagmine_fragMinSpecDiff=25
ptmshepherd.diagmine_minIonsPerSpec=2
ptmshepherd.diagmine_minPeps=25
ptmshepherd.diagmine_mode=false
ptmshepherd.diagmine_pepMinFoldChange=3.0
ptmshepherd.diagmine_pepMinSpecDiff=25
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=2,0.5,0.1
ptmshepherd.prob_dhexY=2,0.5
ptmshepherd.prob_neuacOx=2,0.05,0.2
ptmshepherd.prob_neugcOx=2,0.05,0.2
ptmshepherd.prob_phosphoOx=2,0.05,0.2
ptmshepherd.prob_regY=5,0.5
ptmshepherd.prob_sulfoOx=2,0.05,0.2
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
saintexpress.fragpipe.cmd-opts=
saintexpress.max-replicates=10
saintexpress.run-saint-express=false
saintexpress.virtual-controls=100
speclibgen.easypqp.extras.max_delta_ppm=15
speclibgen.easypqp.extras.max_delta_unimod=0.02
speclibgen.easypqp.extras.rt_lowess_fraction=0
speclibgen.easypqp.fragment.a=false
speclibgen.easypqp.fragment.b=true
speclibgen.easypqp.fragment.c=false
speclibgen.easypqp.fragment.x=false
speclibgen.easypqp.fragment.y=true
speclibgen.easypqp.fragment.z=false
speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM

speclibgen.easypqp.neutral_loss=false
speclibgen.easypqp.rt-cal=noiRT
speclibgen.easypqp.select-file.text=
speclibgen.easypqp.select-im-file.text=
speclibgen.keep-intermediate-files=false
speclibgen.run-speclibgen=true
tab-run.delete_calibrated_mzml=false
tab-run.delete_temp_files=false
tmtintegrator.add_Ref=-1
tmtintegrator.aggregation_method=0
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=TMT-6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.glyco_qval=-1
tmtintegrator.groupby=0
tmtintegrator.log2transformed=true
tmtintegrator.max_pep_prob_thres=0
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=Bridge
tmtintegrator.run-tmtintegrator=false
tmtintegrator.tolerance=20
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=0
tmtintegrator.unique_pep=false
tmtintegrator.use_glycan_composition=false
workdir=D\:\\17915\\Temp
workflow.input.data-type.im-ms=false
workflow.input.data-type.regular-ms=true
workflow.ram=0
workflow.threads=63

CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.mzML 63 Done in 6225 ms. Process 'CheckCentroid' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck INFO[15:56:58] Executing Workspace v4.8.1
INFO[15:56:58] Removing workspace
WARN[15:56:58] Cannot read file. open .meta\meta.bin: The system cannot find the path specified. INFO[15:56:58] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\e92b1203-0a35-4118-baee-77b879b2cf05 INFO[15:56:59] Executing Workspace v4.8.1
INFO[15:56:59] Creating workspace
INFO[15:56:59] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx844G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.mzML MSFragger version MSFragger-3.7 Batmass-IO version 1.28.9 timsdata library version timsdata-2-21-0-4 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. timdTOF .d reading tool. Copyright (c) 2022 by Bruker Daltonics GmbH & Co. KG. All rights reserved. System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK JVM started with 844 GB memory Checking database... Checking spectral files... D:\17915\FS1_MB_17915_2_29012023.mzML: Scans = 84704 ***FIRST SEARCH**** Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 5 minimum_ratio = 0.0 intensity_transform = 0 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.10 Da. 344926862 fragments to be searched in 1 slices (5.14 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.63 s

FS1_MB_17915_2_29012023.mzML 10.0 s | deisotoping 5.1 s [progress: 84552/84552 (100%) - 10998 spectra/s] 7.7s | postprocessing 0.8 s ***FIRST SEARCH DONE IN 0.889 MIN**

***MASS CALIBRATION AND PARAMETER OPTIMIZATION* -----	---------------	---------------	---------------	---------------
Run	Median MAD	Median MAD	Median MAD	Median MAD
001	Not enough data to perform mass calibration. Using the uncalibrated data.
-----	---------------	---------------	---------------	---------------

Finding the optimal parameters: -------|-------|-------|-------|-------|-------|-------|-------|-------

MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50

-------|-------|-------|-------|-------|-------|-------|-------|-------

Count	4	29	33	29	skip rest

-------	-------	-------	-------	-------	-------	------- Peaks
Count	33	44	51	51	41	skip rest
-------	-------	-------	-------	-------	-------	-------
-------	-------
Int.	1
-------	-------
Count	49
-------	-------
-------	-------
Rm P.	0
-------	-------
Count	51
-------	-------

New fragment_mass_tolerance = 10 PPM New use_topN_peaks = 150 New minimum_ratio = 0.010000 New intensity_transform = 0 New remove_precursor_peak = 0 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 3.195 MIN*****

****MAIN SEARCH**** Checking database... Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false isotope_error = 0/1/2 mass_offsets = 0.0 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 150 minIonsScoring = 2 min_matched_fragments = 5 minimum_ratio = 0.01 intensity_transform = 0 activation_types = all remove_precursor_peak = 0 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 variable_mod_05 = -18.0106 nE 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.05 Da. 368492928 fragments to be searched in 1 slices (5.49 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.83 s

FS1_MB_17915_2_29012023.mzML 6.1 s | deisotoping 2.0 s [progress: 84552/84552 (100%) - 49101 spectra/s] 1.7s | remapping alternative proteins and postprocessing 3.7 s MAIN SEARCH DONE IN 0.382 MIN

***TOTAL TIME 4.467 MIN**** Process 'MSFragger' finished, exit code: 0 MSFragger move pepxml C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pepXML D:\17915\Temp\FS1_MB_17915_2_29012023.pepXML Process 'MSFragger move pepxml' finished, exit code: 0 MSFragger move pin C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pin D:\17915\Temp\FS1_MB_17915_2_29012023.pin Process 'MSFragger move pin' finished, exit code: 0 MSBooster [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\msbooster-1.1.11.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" Features.MainClass --paramsList D:\17915\Temp\msbooster_params.txt MSBooster v1.1.11 Using 63 threads Generating input file for DIA-NN 2497 unique peptides from 8558 PSMs Writing DIA-NN input file Diann input file generation took 742 milliseconds Input file at D:\17915\Temp\spectraRT.tsv 2497 unique peptides from 8558 PSMs createFull input file generation took 97 milliseconds Input file at D:\17915\Temp\spectraRT_full.tsv Generating DIA-NN predictions C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\diann\1.8.2_beta_8\win\DiaNN.exe --lib D:\17915\Temp\spectraRT.tsv --predict --threads 63 --strip-unknown-mods --mod TMT,229.1629 --predict-n-frag 100 DIA-NN 1.8.2 beta 8 (Data-Independent Acquisition by Neural Networks) Compiled on Sep 15 2022 18:28:57 Current date and time: Mon Feb 6 16:01:33 2023 CPU: AuthenticAMD AMD EPYC 7552 48-Core Processor SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2 SSE4a Logical CPU cores: 64 Predicted spectra will be saved in a binary format Thread number set to 63 DIA-NN will use deep learning to predict spectra/RTs/IMs even for peptides carrying modifications which are not recognised by the deep learning predictor. In this scenario, if also generating a spectral library from the DIA data or using the MBR mode, it might or might not be better (depends on the data) to also use the --out-measured-rt option - it's recommended to test it with and without this option Modification TMT with mass delta 229.163 added to the list of recognised modifications for spectral library-based search Deep learning predictor will predict 100 fragments Cannot find a UniMod modification match for TMT: 73.0618 minimal mass discrepancy; using the original modificaiton name

0 files will be processed [0:00] Loading spectral library D:\17915\Temp\spectraRT.tsv [0:00] Finding proteotypic peptides (assuming that the list of UniProt ids provided for each peptide is complete) [0:00] Spectral library loaded: 0 protein isoforms, 0 protein groups and 2497 precursors in 2497 elution groups. [0:00] Encoding peptides for spectra and RTs prediction [0:00] Predicting spectra and IMs [0:05] Predicting RTs [0:07] Decoding predicted spectra and IMs [0:07] Decoding RTs [0:07] Saving the list of predictions to D:\17915\Temp\spectraRT.predicted.bin Finished Done generating DIA-NN predictions Model running took 8217 milliseconds Generating edited pin with following features: [unweightedSpectralEntropy, deltaRTLOESS] Loading predicted spectra Processing FS1_MB_17915_2_29012023_uncalibrated.mzML RT regression using 4 PSMs Edited pin file at D:\17915\Temp\FS1_MB_17915_2_29012023_edited.pin Done in 13476 ms Process 'MSBooster' finished, exit code: 0 Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator version 3.05.0, Build Date May 18 2020 02:35:01 Copyright (c) 2006-9 University of Washington. All rights reserved. Written by Lukas Käll @.***) in the Department of Genome Sciences at the University of Washington. Issued command: C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Started Mon Feb 6 16:01:54 2023 Hyperparameters: selectionFdr=0.01, Cpos=0, Cneg=0, maxNiter=10

Reading tab-delimited input from datafile FS1_MB_17915_2_29012023_edited.pin Features: retentiontime rank abs_ppm isotope_errors log10_evalue hyperscore delta_hyperscore matched_ion_num complementary_ions ion_series weighted_average_abs_fragment_ppm peptide_length ntt nmc charge_1 charge_2 charge_3 charge_4 charge_5 charge_6 charge_7_or_more -17.0265nQnC -18.0106nE 15.9949M unweighted_spectral_entropy delta_RT_loess Found 8558 PSMs Concatenated search input detected and --post-processing-tdc flag set. Applying target-decoy competition on Percolator scores. Train/test set contains 4278 positives and 4280 negatives, size ratio=0.999533 and pi0=1 Selecting Cpos by cross-validation. Selecting Cneg by cross-validation. Split 1: Selected feature 6 as initial direction. Could separate 34 training set positives with q<0.01 in that direction. Split 2: Selected feature 5 as initial direction. Could separate 35 training set positives with q<0.01 in that direction. Split 3: Selected feature 5 as initial direction. Could separate 28 training set positives with q<0.01 in that direction. Found 0 test set positives with q<0.01 in initial direction Reading in data and feature calculation took 0.483 cpu seconds or 1 seconds wall clock time. ---Training with Cpos selected by cross validation, Cneg selected by cross validation, initial_fdr=0.01, fdr=0.01 Iteration 1: Estimated 0 PSMs with q<0.01 Iteration 2: Estimated 59 PSMs with q<0.01 Iteration 3: Estimated 72 PSMs with q<0.01 Iteration 4: Estimated 181 PSMs with q<0.01 Iteration 5: Estimated 188 PSMs with q<0.01 Iteration 6: Estimated 190 PSMs with q<0.01 Iteration 7: Estimated 191 PSMs with q<0.01 Iteration 8: Estimated 194 PSMs with q<0.01 Iteration 9: Estimated 200 PSMs with q<0.01 Iteration 10: Estimated 203 PSMs with q<0.01 Learned normalized SVM weights for the 3 cross-validation splits: Split1 Split2 Split3 FeatureName -4.4913 0.2390 -0.2953 retentiontime 0.0000 0.0000 0.0000 rank 1.0970 1.3365 0.6797 abs_ppm 5.2691 1.9916 2.6877 isotope_errors -2.9522 -3.6102 -3.3298 log10_evalue 0.8751 0.0799 0.7274 hyperscore -0.7880 -0.4236 0.0302 delta_hyperscore -0.8479 -1.4030 -1.6929 matched_ion_num 0.7424 0.1502 -0.4959 complementary_ions 1.7124 1.3828 1.5140 ion_series 0.3132 -0.1346 0.1116 weighted_average_abs_fragment_ppm -0.1715 -0.6610 -0.4867 peptide_length 0.0000 0.0000 0.0000 ntt -0.7360 -1.1251 -1.0992 nmc 0.0000 0.0000 0.0000 charge_1 0.0000 0.0000 0.0000 charge_2 0.0000 0.0000 0.0000 charge_3 0.0000 0.0000 0.0000 charge_4 0.0000 0.0000 0.0000 charge_5 0.0000 0.0000 0.0000 charge_6 0.0000 0.0000 0.0000 charge_7_or_more 0.7435 -0.1741 -0.0229 -17.0265nQnC 0.0273 0.9277 0.5164 -18.0106nE 1.2156 2.1702 1.5007 15.9949M 0.7971 1.4475 0.9711 unweighted_spectral_entropy -6.1394 -3.3990 -3.6452 delta_RT_loess -13.0417 -12.0309 -11.9982 m0 Found 0 test set PSMs with q<0.01. No targets found with q<0.01 Resetting score vector, using default vector. Use --override flag to prevent this. Split 1: Selected feature 6 as initial direction. Could separate 34 training set positives with q<0.01 in that direction. Split 2: Selected feature 5 as initial direction. Could separate 35 training set positives with q<0.01 in that direction. Split 3: Selected feature 5 as initial direction. Could separate 28 training set positives with q<0.01 in that direction. Selected best-scoring PSM per scan+expMass (target-decoy competition): 4278 target PSMs and 4280 decoy PSMs. Calculating q values. Final list yields 0 target PSMs with q<0.01. Calculating posterior error probabilities (PEPs). Processing took 10.89 cpu seconds or 11 seconds wall clock time. Process 'Percolator' finished, exit code: 0 Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Process 'Percolator: Convert to pepxml' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt INFO[16:02:09] Executing ProteinProphet v4.8.1
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 D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml... ...read in 0 1+, 0 2+, 18 3+, 0 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.5

Initializing 4 peptide weights: 0%...50%...100% Calculating protein lengths and molecular weights from database D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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 .........:.........:.........:.........:.........:.........:.........:.........:....... Total: 40874 Computing degenerate peptides for 4 proteins: 0%...50%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Calculating sensitivity...and error tables... INFO: mu=6.78873e-09, db_size=45578978 Computing MU for 4 proteins: 0%...50%...100% INFO[16:02:12] Done
Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev INFO[16:02:12] Executing Database v4.8.1
INFO[16:02:12] Annotating the database
INFO[16:02:23] Done
Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor INFO[16:02:24] Executing Filter v4.8.1
INFO[16:02:24] Processing peptide identification files
INFO[16:02:24] Parsing D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml INFO[16:02:24] 1+ Charge profile decoy=0 target=0 INFO[16:02:24] 2+ Charge profile decoy=0 target=0 INFO[16:02:24] 3+ Charge profile decoy=0 target=18 INFO[16:02:24] 4+ Charge profile decoy=0 target=0 INFO[16:02:24] 5+ Charge profile decoy=0 target=0 INFO[16:02:24] 6+ Charge profile decoy=0 target=0 INFO[16:02:24] Database search results ions=4 peptides=4 psms=18 INFO[16:02:24] Converged to 0.00 % FDR with 18 PSMs decoy=0 threshold=0.734154 total=18 INFO[16:02:24] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Protein inference results decoy=0 target=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Proteins decoy=0 threshold=0.8053 total=4 INFO[16:02:24] 2D FDR estimation: Protein mirror image decoy=4 target=4 INFO[16:02:24] Second filtering results ions=4 peptides=4 psms=18 INFO[16:02:24] Converged to 0.00 % FDR with 18 PSMs decoy=0 threshold=0.734154 total=18 INFO[16:02:24] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Post processing identifications
INFO[16:02:24] Assigning protein identifications to layers
INFO[16:02:25] Processing protein inference
INFO[16:02:26] Synchronizing PSMs and proteins
INFO[16:02:26] Final report numbers after FDR filtering, and post-processing ions=4 peptides=4 proteins=4 psms=18 INFO[16:02:26] Saving
INFO[16:02:26] Done
Process 'PhilosopherFilter' finished, exit code: 0 PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report INFO[16:02:26] Executing Report v4.8.1
INFO[16:02:26] Creating reports
INFO[16:02:26] Done
Process 'PhilosopherReport' finished, exit code: 0 SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt File list provided Spectral library building Commands to execute: 'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' convert --max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types '['"'"'b'"'"','"'"'y'"'"',]' --enable_unannotated --pepxml 'D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml' --spectra 'D:\17915\FS1_MB_17915_2_29012023.mzML' --exclude-range -1.5,3.5 --psms FS1_MB_17915_2_29012023.psmpkl --peaks FS1_MB_17915_2_29012023.peakpkl

'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' library --psmtsv 'D:\17915\Temp\psm.tsv' --peptidetsv 'D:\17915\Temp\peptide.tsv' --out easypqp_lib_openswath.tsv --rt_lowess_fraction 0.0 'D:\17915\Temp\filelist_easypqp_library.txt'

easypqp, version 0.1.35
Executing ['C:\\Users\\protolab\\Anaconda3\\Scripts\\easypqp.exe', 'convert', '--max_delta_unimod', '0.02', '--max_delta_ppm', '15.0', '--fragment_types', "['b','y',]", '--enable_unannotated', '--pepxml', 'D:\\17915\\Temp\\interact-FS1_MB_17915_2_29012023.pep.xml', '--spectra', 'D:\\17915\\FS1_MB_17915_2_29012023.mzML', '--exclude-range', '-1.5,3.5', '--psms', 'FS1_MB_17915_2_29012023.psmpkl', '--peaks', 'FS1_MB_17915_2_29012023.peakpkl']
Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
    return self._engine.get_loc(casted_key)
  File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
  File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
    return _run_code(code, main_globals, None,
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

    exec(code, run_globals)
  File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
    return self.main(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
    rv = self.invoke(ctx)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
    rv.append(sub_ctx.command.invoke(sub_ctx))
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
    return __callback(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
    generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
    pepida = pepida.loc[np.isfinite(pepida['irt'])]
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
    indexer = self.columns.get_loc(key)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
    raise KeyError(key) from err
KeyError: 'irt'
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 18 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
                 base_name  modified_peptide
0  FS1_MB_17915_2_29012023                 4
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 4.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Not enough peptides could be found for alignment.
Using ciRT for alignment
Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
    return self._engine.get_loc(casted_key)
  File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
  File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
  File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
    return _run_code(code, main_globals, None,
  File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

    exec(code, run_globals)
  File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
    return self.main(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
    rv = self.invoke(ctx)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
    rv.append(sub_ctx.command.invoke(sub_ctx))
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
    return __callback(*args, **kwargs)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
    generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
    pepida = pepida.loc[np.isfinite(pepida['irt'])]
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
    indexer = self.columns.get_loc(key)
  File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
    raise KeyError(key) from err
KeyError: 'irt'
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 18 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Library not generated, not enough peptides could be found for alignment.

Library not generated, not enough peptides could be found for alignment.

Please try using other options for alignment (e.g. ciRT if used other options)
Process 'SpecLibGen' finished, exit code: 1
Process returned non-zero exit code, stopping

Cancelling 2 remaining tasks

Did you change the file type to DIA?

Get Outlook for iOShttps://aka.ms/o0ukef

From: yishai99 @.> Sent: Tuesday, February 7, 2023 1:08:43 AM To: Nesvilab/FragPipe @.> Cc: Nesvizhskii, Alexey @.>; Comment @.> Subject: Re: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

I tried again, this time making sure to Load the right workflow. Still get an error (attached).

On 6 Feb 2023, at 17:58, Alexey Nesvizhskii @.***> wrote:

There is some issue with Github system, so I am responding again in case you did not receive it

You did something wrong. I think you wanted to use DIA-Umpire workflow but did not click LOAD Or maybe you did, but did not annotate the file as DIA type (it is DDA by default).

Also, for full DIA workflow (that includes DIA-NN for quant) I suggest you convert to mzML

Alexey

From: yishai99 @.> Sent: Monday, February 6, 2023 3:08 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0. Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Please try using other options for alignment (e.g. ciRT if used other options) Process 'SpecLibGen' finished, exit code: 1 Process returned non-zero exit code, stopping

— Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/993, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM64MQWJLCR7C5QMW5TLWWCWPVANCNFSM6AAAAAAUSLLM6A. You are receiving this because you are subscribed to this thread.Message ID: @.**@.>>

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System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK .NET Core Info: N/A

Version info: FragPipe version 19.1 MSFragger version 3.7 IonQuant version 1.8.10 Philosopher version 4.8.1

LCMS files: Experiment/Group:

D:\17915\FS1_MB_17915_2_29012023.mzML DDA


Execution order:

Cmd: [START], Work dir: [D:\17915\Temp]
Cmd: [CheckCentroid], Work dir: [D:\17915\Temp]
Cmd: [WorkspaceCleanInit], Work dir: [D:\17915\Temp]
Cmd: [MSFragger], Work dir: [D:\17915\Temp]
Cmd: [MSBooster], Work dir: [D:\17915\Temp]
Cmd: [Percolator], Work dir: [D:\17915\Temp]
Cmd: [ProteinProphet], Work dir: [D:\17915\Temp]
Cmd: [PhilosopherDbAnnotate], Work dir: [D:\17915\Temp]
Cmd: [PhilosopherFilter], Work dir: [D:\17915\Temp]
Cmd: [PhilosopherReport], Work dir: [D:\17915\Temp]
Cmd: [SpecLibGen], Work dir: [D:\17915\Temp]
Cmd: [WorkspaceClean], Work dir: [D:\17915\Temp]


>rev_sp|A0A024RBG1|NUD4B_HUMAN Diphosphoinositol polyphosphate phosphohydrolase NUDT4B OS=Homo sapiens OX=9606 GN=NUDT4B PE=3 SV=1
>rev_sp|O43908|NKG2F_HUMAN NKG2-F type II integral membrane protein OS=Homo sapiens OX=9606 GN=KLRC4 PE=2 SV=2
>rev_sp|P0DPK2|H3Y1_HUMAN Histone H3.Y OS=Homo sapiens OX=9606 GN=H3Y1 PE=1 SV=1
>rev_sp|P50336|PPOX_HUMAN Protoporphyrinogen oxidase OS=Homo sapiens OX=9606 GN=PPOX PE=1 SV=1
>rev_sp|Q14416|GRM2_HUMAN Metabotropic glutamate receptor 2 OS=Homo sapiens OX=9606 GN=GRM2 PE=1 SV=2
>rev_sp|Q6DKK2|TTC19_HUMAN Tetratricopeptide repeat protein 19, mitochondrial OS=Homo sapiens OX=9606 GN=TTC19 PE=1 SV=4
>rev_sp|Q8IX21|SLF2_HUMAN SMC5-SMC6 complex localization factor protein 2 OS=Homo sapiens OX=9606 GN=SLF2 PE=1 SV=2
>rev_sp|Q8WWH5|TRUB1_HUMAN Pseudouridylate synthase TRUB1 OS=Homo sapiens OX=9606 GN=TRUB1 PE=1 SV=1
>rev_sp|Q9BS26|ERP44_HUMAN Endoplasmic reticulum resident protein 44 OS=Homo sapiens OX=9606 GN=ERP44 PE=1 SV=1
>rev_sp|Q9NVH2|INT7_HUMAN Integrator complex subunit 7 OS=Homo sapiens OX=9606 GN=INTS7 PE=1 SV=1
>rev_sp|S4R3P1|HMN13_HUMAN Humanin-like 13 OS=Homo sapiens OX=9606 GN=MTRNR2L13 PE=3 SV=1
>sp|O43869|OR2T1_HUMAN Olfactory receptor 2T1 OS=Homo sapiens OX=9606 GN=OR2T1 PE=3 SV=3
>sp|P0DPF7|TVB63_HUMAN T cell receptor beta variable 6-3 OS=Homo sapiens OX=9606 GN=TRBV6-3 PE=3 SV=1
>sp|P50219|MNX1_HUMAN Motor neuron and pancreas homeobox protein 1 OS=Homo sapiens OX=9606 GN=MNX1 PE=1 SV=3
>sp|Q14390|GGTL2_HUMAN Glutathione hydrolase light chain 2 OS=Homo sapiens OX=9606 GN=GGTLC2 PE=1 SV=4
>sp|Q6DHV7|ADAL_HUMAN Adenosine deaminase-like protein OS=Homo sapiens OX=9606 GN=ADAL PE=1 SV=2
>sp|Q8IX05|CD302_HUMAN CD302 antigen OS=Homo sapiens OX=9606 GN=CD302 PE=1 SV=1
>sp|Q8WWF3|SSMM1_HUMAN Serine-rich single-pass membrane protein 1 OS=Homo sapiens OX=9606 GN=SSMEM1 PE=1 SV=1
>sp|Q9BRX5|PSF3_HUMAN DNA replication complex GINS protein PSF3 OS=Homo sapiens OX=9606 GN=GINS3 PE=1 SV=1
>sp|Q9NVE5|UBP40_HUMAN Ubiquitin carboxyl-terminal hydrolase 40 OS=Homo sapiens OX=9606 GN=USP40 PE=1 SV=3
>sp|Q9Y6Y8|S23IP_HUMAN SEC23-interacting protein OS=Homo sapiens OX=9606 GN=SEC23IP PE=1 SV=1

# FragPipe v19.1ui state cache

# Please edit the following path to point to the correct location.
# In Windows, please replace single '\' with '\\'
database.db-path=D\:\\Databases\\2023-01-25-decoys-contam-Human_SP_Jan2023.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=true
diann.run-specific-protein-q-value=false
diann.unrelated-runs=false
diann.use-predicted-spectra=true
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=true
fragpipe-config.bin-ionquant=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\IonQuant-1.8.10.jar
fragpipe-config.bin-msfragger=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\MSFragger-3.7\\MSFragger-3.7.jar
fragpipe-config.bin-philosopher=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\philosopher_v4.8.1_windows_amd64\\philosopher.exe
fragpipe-config.bin-python=C\:\\Users\\protolab\\Anaconda3\\python.exe

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=10
ionquant.medium=
ionquant.minfreq=0
ionquant.minions=1
ionquant.minisotopes=1
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=true
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=0/1/2
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=5
msfragger.min_sequence_matches=2
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.00
msfragger.misc.fragger.clear-mz-hi=0
msfragger.misc.fragger.clear-mz-lo=0
msfragger.misc.fragger.digest-mass-hi=5000
msfragger.misc.fragger.digest-mass-lo=500
msfragger.misc.fragger.enzyme-dropdown-1=stricttrypsin
msfragger.misc.fragger.enzyme-dropdown-2=null
msfragger.misc.fragger.precursor-charge-hi=4
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=stricttrypsin
msfragger.search_enzyme_name_2=null
msfragger.search_enzyme_nocut_1=
msfragger.search_enzyme_nocut_2=
msfragger.search_enzyme_sense_1=C
msfragger.search_enzyme_sense_2=C
msfragger.table.fix-mods=0.0,C-Term Peptide,true,-1; 0.0,N-Term Peptide,true,-1; 0.0,C-Term Protein,true,-1; 0.0,N-Term Protein,true,-1; 0.0,G (glycine),true,-1; 0.0,A (alanine),true,-1; 0.0,S (serine),true,-1; 0.0,P (proline),true,-1; 0.0,V (valine),true,-1; 0.0,T (threonine),true,-1; 57.02146,C (cysteine),true,-1; 0.0,L (leucine),true,-1; 0.0,I (isoleucine),true,-1; 0.0,N (asparagine),true,-1; 0.0,D (aspartic acid),true,-1; 0.0,Q (glutamine),true,-1; 0.0,K (lysine),true,-1; 0.0,E (glutamic acid),true,-1; 0.0,M (methionine),true,-1; 0.0,H (histidine),true,-1; 0.0,F (phenylalanine),true,-1; 0.0,R (arginine),true,-1; 0.0,Y (tyrosine),true,-1; 0.0,W (tryptophan),true,-1; 0.0,B ,true,-1; 0.0,J,true,-1; 0.0,O,true,-1; 0.0,U,true,-1; 0.0,X,true,-1; 0.0,Z,true,-1
msfragger.table.var-mods=15.9949,M,true,3; 42.0106,[^,true,1; 79.96633,STY,false,3; -17.0265,nQnC,true,1; -18.0106,nE,true,1; 0.0,site_06,false,1; 0.0,site_07,false,1; 0.0,site_08,false,1; 0.0,site_09,false,1; 0.0,site_10,false,1; 0.0,site_11,false,1; 0.0,site_12,false,1; 0.0,site_13,false,1; 0.0,site_14,false,1; 0.0,site_15,false,1; 0.0,site_16,false,1
msfragger.track_zero_topN=0
msfragger.use_all_mods_in_first_search=false
msfragger.use_topN_peaks=300
msfragger.write_calibrated_mzml=false
msfragger.write_uncalibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
opair.activation1=HCD
opair.activation2=ETD
opair.glyco_db=
opair.max_glycans=4
opair.max_isotope_error=2
opair.min_isotope_error=0
opair.ms1_tol=20
opair.ms2_tol=20
opair.reverse_scan_order=false
opair.run-opair=false
opair.single_scan_type=false
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=false
percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc

percolator.keep-tsv-files=false
percolator.min-prob=0.7
percolator.run-percolator=true
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--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=true
phi-report.remove-contaminants=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.cap_y_ions=
ptmshepherd.decoy_type=1
ptmshepherd.diag_ions=
ptmshepherd.diagextract_mode=false
ptmshepherd.diagmine_diagMinFoldChange=3.0
ptmshepherd.diagmine_diagMinSpecDiff=25
ptmshepherd.diagmine_fragMinFoldChange=3.0
ptmshepherd.diagmine_fragMinPropensity=12.5
ptmshepherd.diagmine_fragMinSpecDiff=25
ptmshepherd.diagmine_minIonsPerSpec=2
ptmshepherd.diagmine_minPeps=25
ptmshepherd.diagmine_mode=false
ptmshepherd.diagmine_pepMinFoldChange=3.0
ptmshepherd.diagmine_pepMinSpecDiff=25
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=2,0.5,0.1
ptmshepherd.prob_dhexY=2,0.5
ptmshepherd.prob_neuacOx=2,0.05,0.2
ptmshepherd.prob_neugcOx=2,0.05,0.2
ptmshepherd.prob_phosphoOx=2,0.05,0.2
ptmshepherd.prob_regY=5,0.5
ptmshepherd.prob_sulfoOx=2,0.05,0.2
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
saintexpress.fragpipe.cmd-opts=
saintexpress.max-replicates=10
saintexpress.run-saint-express=false
saintexpress.virtual-controls=100
speclibgen.easypqp.extras.max_delta_ppm=15
speclibgen.easypqp.extras.max_delta_unimod=0.02
speclibgen.easypqp.extras.rt_lowess_fraction=0
speclibgen.easypqp.fragment.a=false
speclibgen.easypqp.fragment.b=true
speclibgen.easypqp.fragment.c=false
speclibgen.easypqp.fragment.x=false
speclibgen.easypqp.fragment.y=true
speclibgen.easypqp.fragment.z=false
speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM

speclibgen.easypqp.neutral_loss=false
speclibgen.easypqp.rt-cal=noiRT
speclibgen.easypqp.select-file.text=
speclibgen.easypqp.select-im-file.text=
speclibgen.keep-intermediate-files=false
speclibgen.run-speclibgen=true
tab-run.delete_calibrated_mzml=false
tab-run.delete_temp_files=false
tmtintegrator.add_Ref=-1
tmtintegrator.aggregation_method=0
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=TMT-6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.glyco_qval=-1
tmtintegrator.groupby=0
tmtintegrator.log2transformed=true
tmtintegrator.max_pep_prob_thres=0
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=Bridge
tmtintegrator.run-tmtintegrator=false
tmtintegrator.tolerance=20
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=0
tmtintegrator.unique_pep=false
tmtintegrator.use_glycan_composition=false
workdir=D\:\\17915\\Temp
workflow.input.data-type.im-ms=false
workflow.input.data-type.regular-ms=true
workflow.ram=0
workflow.threads=63

CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.mzML 63 Done in 6225 ms. Process 'CheckCentroid' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck INFO[15:56:58] Executing Workspace v4.8.1 INFO[15:56:58] Removing workspace WARN[15:56:58] Cannot read file. open .meta\meta.bin: The system cannot find the path specified. INFO[15:56:58] Done Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\e92b1203-0a35-4118-baee-77b879b2cf05 INFO[15:56:59] Executing Workspace v4.8.1 INFO[15:56:59] Creating workspace INFO[15:56:59] Done Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx844G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.mzML MSFragger version MSFragger-3.7 Batmass-IO version 1.28.9 timsdata library version timsdata-2-21-0-4 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. timdTOF .d reading tool. Copyright (c) 2022 by Bruker Daltonics GmbH & Co. KG. All rights reserved. System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK JVM started with 844 GB memory Checking database... Checking spectral files... D:\17915\FS1_MB_17915_2_29012023.mzML: Scans = 84704 ***FIRST SEARCH**** Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 5 minimum_ratio = 0.0 intensity_transform = 0 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.10 Da. 344926862 fragments to be searched in 1 slices (5.14 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.63 s

FS1_MB_17915_2_29012023.mzML 10.0 s | deisotoping 5.1 s [progress: 84552/84552 (100%) - 10998 spectra/s] 7.7s | postprocessing 0.8 s ***FIRST SEARCH DONE IN 0.889 MIN**

***MASS CALIBRATION AND PARAMETER OPTIMIZATION* -----	---------------	---------------	---------------	---------------
Run	Median MAD	Median MAD	Median MAD	Median MAD
001	Not enough data to perform mass calibration. Using the uncalibrated data.
-----	---------------	---------------	---------------	---------------

Finding the optimal parameters: -------|-------|-------|-------|-------|-------|-------|-------|-------

MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50

-------|-------|-------|-------|-------|-------|-------|-------|-------

Count	4	29	33	29	skip rest

-------	-------	-------	-------	-------	-------	------- Peaks
Count	33	44	51	51	41	skip rest
-------	-------	-------	-------	-------	-------	-------
-------	-------
Int.	1
-------	-------
Count	49
-------	-------
-------	-------
Rm P.	0
-------	-------
Count	51
-------	-------

****MAIN SEARCH**** Checking database... Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false isotope_error = 0/1/2 mass_offsets = 0.0 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 150 minIonsScoring = 2 min_matched_fragments = 5 minimum_ratio = 0.01 intensity_transform = 0 activation_types = all remove_precursor_peak = 0 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 variable_mod_05 = -18.0106 nE 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.05 Da. 368492928 fragments to be searched in 1 slices (5.49 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.83 s

FS1_MB_17915_2_29012023.mzML 6.1 s | deisotoping 2.0 s [progress: 84552/84552 (100%) - 49101 spectra/s] 1.7s | remapping alternative proteins and postprocessing 3.7 s MAIN SEARCH DONE IN 0.382 MIN

0 files will be processed [0:00] Loading spectral library D:\17915\Temp\spectraRT.tsv [0:00] Finding proteotypic peptides (assuming that the list of UniProt ids provided for each peptide is complete) [0:00] Spectral library loaded: 0 protein isoforms, 0 protein groups and 2497 precursors in 2497 elution groups. [0:00] Encoding peptides for spectra and RTs prediction [0:00] Predicting spectra and IMs [0:05] Predicting RTs [0:07] Decoding predicted spectra and IMs [0:07] Decoding RTs [0:07] Saving the list of predictions to D:\17915\Temp\spectraRT.predicted.bin Finished Done generating DIA-NN predictions Model running took 8217 milliseconds Generating edited pin with following features: [unweightedSpectralEntropy, deltaRTLOESS] Loading predicted spectra Processing FS1_MB_17915_2_29012023_uncalibrated.mzML RT regression using 4 PSMs Edited pin file at D:\17915\Temp\FS1_MB_17915_2_29012023_edited.pin Done in 13476 ms Process 'MSBooster' finished, exit code: 0 Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator version 3.05.0, Build Date May 18 2020 02:35:01 Copyright (c) 2006-9 University of Washington. All rights reserved. Written by Lukas Käll @.***) in the Department of Genome Sciences at the University of Washington. Issued command: C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Started Mon Feb 6 16:01:54 2023 Hyperparameters: selectionFdr=0.01, Cpos=0, Cneg=0, maxNiter=10

Reading tab-delimited input from datafile FS1_MB_17915_2_29012023_edited.pin Features: retentiontime rank abs_ppm isotope_errors log10_evalue hyperscore delta_hyperscore matched_ion_num complementary_ions ion_series weighted_average_abs_fragment_ppm peptide_length ntt nmc charge_1 charge_2 charge_3 charge_4 charge_5 charge_6 charge_7_or_more -17.0265nQnC -18.0106nE 15.9949M unweighted_spectral_entropy delta_RT_loess Found 8558 PSMs Concatenated search input detected and --post-processing-tdc flag set. Applying target-decoy competition on Percolator scores. Train/test set contains 4278 positives and 4280 negatives, size ratio=0.999533 and pi0=1 Selecting Cpos by cross-validation. Selecting Cneg by cross-validation. Split 1: Selected feature 6 as initial direction. Could separate 34 training set positives with q<0.01 in that direction. Split 2: Selected feature 5 as initial direction. Could separate 35 training set positives with q<0.01 in that direction. Split 3: Selected feature 5 as initial direction. Could separate 28 training set positives with q<0.01 in that direction. Found 0 test set positives with q<0.01 in initial direction Reading in data and feature calculation took 0.483 cpu seconds or 1 seconds wall clock time. ---Training with Cpos selected by cross validation, Cneg selected by cross validation, initial_fdr=0.01, fdr=0.01 Iteration 1: Estimated 0 PSMs with q<0.01 Iteration 2: Estimated 59 PSMs with q<0.01 Iteration 3: Estimated 72 PSMs with q<0.01 Iteration 4: Estimated 181 PSMs with q<0.01 Iteration 5: Estimated 188 PSMs with q<0.01 Iteration 6: Estimated 190 PSMs with q<0.01 Iteration 7: Estimated 191 PSMs with q<0.01 Iteration 8: Estimated 194 PSMs with q<0.01 Iteration 9: Estimated 200 PSMs with q<0.01 Iteration 10: Estimated 203 PSMs with q<0.01 Learned normalized SVM weights for the 3 cross-validation splits: Split1 Split2 Split3 FeatureName -4.4913 0.2390 -0.2953 retentiontime 0.0000 0.0000 0.0000 rank 1.0970 1.3365 0.6797 abs_ppm 5.2691 1.9916 2.6877 isotope_errors -2.9522 -3.6102 -3.3298 log10_evalue 0.8751 0.0799 0.7274 hyperscore -0.7880 -0.4236 0.0302 delta_hyperscore -0.8479 -1.4030 -1.6929 matched_ion_num 0.7424 0.1502 -0.4959 complementary_ions 1.7124 1.3828 1.5140 ion_series 0.3132 -0.1346 0.1116 weighted_average_abs_fragment_ppm -0.1715 -0.6610 -0.4867 peptide_length 0.0000 0.0000 0.0000 ntt -0.7360 -1.1251 -1.0992 nmc 0.0000 0.0000 0.0000 charge_1 0.0000 0.0000 0.0000 charge_2 0.0000 0.0000 0.0000 charge_3 0.0000 0.0000 0.0000 charge_4 0.0000 0.0000 0.0000 charge_5 0.0000 0.0000 0.0000 charge_6 0.0000 0.0000 0.0000 charge_7_or_more 0.7435 -0.1741 -0.0229 -17.0265nQnC 0.0273 0.9277 0.5164 -18.0106nE 1.2156 2.1702 1.5007 15.9949M 0.7971 1.4475 0.9711 unweighted_spectral_entropy -6.1394 -3.3990 -3.6452 delta_RT_loess -13.0417 -12.0309 -11.9982 m0 Found 0 test set PSMs with q<0.01. No targets found with q<0.01 Resetting score vector, using default vector. Use --override flag to prevent this. Split 1: Selected feature 6 as initial direction. Could separate 34 training set positives with q<0.01 in that direction. Split 2: Selected feature 5 as initial direction. Could separate 35 training set positives with q<0.01 in that direction. Split 3: Selected feature 5 as initial direction. Could separate 28 training set positives with q<0.01 in that direction. Selected best-scoring PSM per scan+expMass (target-decoy competition): 4278 target PSMs and 4280 decoy PSMs. Calculating q values. Final list yields 0 target PSMs with q<0.01. Calculating posterior error probabilities (PEPs). Processing took 10.89 cpu seconds or 11 seconds wall clock time. Process 'Percolator' finished, exit code: 0 Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Process 'Percolator: Convert to pepxml' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt INFO[16:02:09] Executing ProteinProphet v4.8.1 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 D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml... ...read in 0 1+, 0 2+, 18 3+, 0 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.5

Initializing 4 peptide weights: 0%...50%...100% Calculating protein lengths and molecular weights from database D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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 .........:.........:.........:.........:.........:.........:.........:.........:....... Total: 40874 Computing degenerate peptides for 4 proteins: 0%...50%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Calculating sensitivity...and error tables... INFO: mu=6.78873e-09, db_size=45578978 Computing MU for 4 proteins: 0%...50%...100% INFO[16:02:12] Done Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev INFO[16:02:12] Executing Database v4.8.1 INFO[16:02:12] Annotating the database INFO[16:02:23] Done Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor INFO[16:02:24] Executing Filter v4.8.1 INFO[16:02:24] Processing peptide identification files INFO[16:02:24] Parsing D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml INFO[16:02:24] 1+ Charge profile decoy=0 target=0 INFO[16:02:24] 2+ Charge profile decoy=0 target=0 INFO[16:02:24] 3+ Charge profile decoy=0 target=18 INFO[16:02:24] 4+ Charge profile decoy=0 target=0 INFO[16:02:24] 5+ Charge profile decoy=0 target=0 INFO[16:02:24] 6+ Charge profile decoy=0 target=0 INFO[16:02:24] Database search results ions=4 peptides=4 psms=18 INFO[16:02:24] Converged to 0.00 % FDR with 18 PSMs decoy=0 threshold=0.734154 total=18 INFO[16:02:24] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Protein inference results decoy=0 target=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Proteins decoy=0 threshold=0.8053 total=4 INFO[16:02:24] 2D FDR estimation: Protein mirror image decoy=4 target=4 INFO[16:02:24] Second filtering results ions=4 peptides=4 psms=18 INFO[16:02:24] Converged to 0.00 % FDR with 18 PSMs decoy=0 threshold=0.734154 total=18 INFO[16:02:24] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Post processing identifications INFO[16:02:24] Assigning protein identifications to layers INFO[16:02:25] Processing protein inference INFO[16:02:26] Synchronizing PSMs and proteins INFO[16:02:26] Final report numbers after FDR filtering, and post-processing ions=4 peptides=4 proteins=4 psms=18 INFO[16:02:26] Saving INFO[16:02:26] Done Process 'PhilosopherFilter' finished, exit code: 0 PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report INFO[16:02:26] Executing Report v4.8.1 INFO[16:02:26] Creating reports INFO[16:02:26] Done Process 'PhilosopherReport' finished, exit code: 0 SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt File list provided Spectral library building Commands to execute: 'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' convert --max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types '['"'"'b'"'"','"'"'y'"'"',]' --enable_unannotated --pepxml 'D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml' --spectra 'D:\17915\FS1_MB_17915_2_29012023.mzML' --exclude-range -1.5,3.5 --psms FS1_MB_17915_2_29012023.psmpkl --peaks FS1_MB_17915_2_29012023.peakpkl

easypqp, version 0.1.35
Executing ['C:\\Users\\protolab\\Anaconda3\\Scripts\\easypqp.exe', 'convert', '--max_delta_unimod', '0.02', '--max_delta_ppm', '15.0', '--fragment_types', "['b','y',]", '--enable_unannotated', '--pepxml', 'D:\\17915\\Temp\\interact-FS1_MB_17915_2_29012023.pep.xml', '--spectra', 'D:\\17915\\FS1_MB_17915_2_29012023.mzML', '--exclude-range', '-1.5,3.5', '--psms', 'FS1_MB_17915_2_29012023.psmpkl', '--peaks', 'FS1_MB_17915_2_29012023.peakpkl']
Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
return self._engine.get_loc(casted_key)
File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
return _run_code(code, main_globals, None,
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

exec(code, run_globals)
File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
return self.main(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
rv = self.invoke(ctx)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
rv.append(sub_ctx.command.invoke(sub_ctx))
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
return __callback(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
pepida = pepida.loc[np.isfinite(pepida['irt'])]
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
indexer = self.columns.get_loc(key)
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
raise KeyError(key) from err
KeyError: 'irt'
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 18 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
base_name modified_peptide
0 FS1_MB_17915_2_29012023 4
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 4.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Not enough peptides could be found for alignment.
Using ciRT for alignment
Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
return self._engine.get_loc(casted_key)
File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
return _run_code(code, main_globals, None,
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

exec(code, run_globals)
File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
return self.main(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
rv = self.invoke(ctx)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
rv.append(sub_ctx.command.invoke(sub_ctx))
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
return __callback(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
pepida = pepida.loc[np.isfinite(pepida['irt'])]
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
indexer = self.columns.get_loc(key)
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
raise KeyError(key) from err
KeyError: 'irt'
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 18 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Library not generated, not enough peptides could be found for alignment.

Library not generated, not enough peptides could be found for alignment.

Please try using other options for alignment (e.g. ciRT if used other options)
Process 'SpecLibGen' finished, exit code: 1
Process returned non-zero exit code, stopping

Cancelling 2 remaining tasks

— Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/993#issuecomment-1420249293, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM65JKMHS7XQS4NYBFFTWWHRGXANCNFSM6AAAAAAUSLLM6A. You are receiving this because you commented.Message ID: @.***>

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Oops. I thought it recognizes it automatically. Now it works, thanks!

On 7 Feb 2023, at 13:45, Alexey Nesvizhskii @.***> wrote:

Did you change the file type to DIA?

Get Outlook for iOShttps://aka.ms/o0ukef

From: yishai99 @.> Sent: Tuesday, February 7, 2023 1:08:43 AM To: Nesvilab/FragPipe @.> Cc: Nesvizhskii, Alexey @.>; Comment @.> Subject: Re: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

I tried again, this time making sure to Load the right workflow. Still get an error (attached).

Y

On 6 Feb 2023, at 17:58, Alexey Nesvizhskii @.***> wrote:

There is some issue with Github system, so I am responding again in case you did not receive it

You did something wrong. I think you wanted to use DIA-Umpire workflow but did not click LOAD Or maybe you did, but did not annotate the file as DIA type (it is DDA by default).

Also, for full DIA workflow (that includes DIA-NN for quant) I suggest you convert to mzML

Alexey

From: yishai99 @.> Sent: Monday, February 6, 2023 3:08 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Error when running DIA_DIA-Umpire_SpecLib_Quant workflow (Issue #993)

External Email - Use Caution

Hi, I tried running one DIA raw file with DIA_DIA-Umpire_SpecLib_Quant and got the error below. I processed the file with Spectronaut and it completed fine. Thanks,

Yishai

Info: 31 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0. Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Library not generated, not enough peptides could be found for alignment. Please try using other options for alignment (e.g. ciRT if used other options) Process 'SpecLibGen' finished, exit code: 1 Process returned non-zero exit code, stopping

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System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK .NET Core Info: N/A

Version info: FragPipe version 19.1 MSFragger version 3.7 IonQuant version 1.8.10 Philosopher version 4.8.1

LCMS files: Experiment/Group:

D:\17915\FS1_MB_17915_2_29012023.mzML DDA

17 commands to execute: CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.mzML 63 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\e92b1203-0a35-4118-baee-77b879b2cf05 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx844G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.mzML MSFragger move pepxml C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pepXML D:\17915\Temp\FS1_MB_17915_2_29012023.pepXML MSFragger move pin C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pin D:\17915\Temp\FS1_MB_17915_2_29012023.pin MSBooster [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\msbooster-1.1.11.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" Features.MainClass --paramsList D:\17915\Temp\msbooster_params.txt Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt WorkspaceClean [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck
Execution order:

Cmd: [START], Work dir: [D:\17915\Temp]
Cmd: [CheckCentroid], Work dir: [D:\17915\Temp]
Cmd: [WorkspaceCleanInit], Work dir: [D:\17915\Temp]
Cmd: [MSFragger], Work dir: [D:\17915\Temp]
Cmd: [MSBooster], Work dir: [D:\17915\Temp]
Cmd: [Percolator], Work dir: [D:\17915\Temp]
Cmd: [ProteinProphet], Work dir: [D:\17915\Temp]
Cmd: [PhilosopherDbAnnotate], Work dir: [D:\17915\Temp]
Cmd: [PhilosopherFilter], Work dir: [D:\17915\Temp]
Cmd: [PhilosopherReport], Work dir: [D:\17915\Temp]
Cmd: [SpecLibGen], Work dir: [D:\17915\Temp]
Cmd: [WorkspaceClean], Work dir: [D:\17915\Temp]
>rev_sp|A0A024RBG1|NUD4B_HUMAN Diphosphoinositol polyphosphate phosphohydrolase NUDT4B OS=Homo sapiens OX=9606 GN=NUDT4B PE=3 SV=1
>rev_sp|O43908|NKG2F_HUMAN NKG2-F type II integral membrane protein OS=Homo sapiens OX=9606 GN=KLRC4 PE=2 SV=2
>rev_sp|P0DPK2|H3Y1_HUMAN Histone H3.Y OS=Homo sapiens OX=9606 GN=H3Y1 PE=1 SV=1
>rev_sp|P50336|PPOX_HUMAN Protoporphyrinogen oxidase OS=Homo sapiens OX=9606 GN=PPOX PE=1 SV=1
>rev_sp|Q14416|GRM2_HUMAN Metabotropic glutamate receptor 2 OS=Homo sapiens OX=9606 GN=GRM2 PE=1 SV=2
>rev_sp|Q6DKK2|TTC19_HUMAN Tetratricopeptide repeat protein 19, mitochondrial OS=Homo sapiens OX=9606 GN=TTC19 PE=1 SV=4
>rev_sp|Q8IX21|SLF2_HUMAN SMC5-SMC6 complex localization factor protein 2 OS=Homo sapiens OX=9606 GN=SLF2 PE=1 SV=2
>rev_sp|Q8WWH5|TRUB1_HUMAN Pseudouridylate synthase TRUB1 OS=Homo sapiens OX=9606 GN=TRUB1 PE=1 SV=1
>rev_sp|Q9BS26|ERP44_HUMAN Endoplasmic reticulum resident protein 44 OS=Homo sapiens OX=9606 GN=ERP44 PE=1 SV=1
>rev_sp|Q9NVH2|INT7_HUMAN Integrator complex subunit 7 OS=Homo sapiens OX=9606 GN=INTS7 PE=1 SV=1
>rev_sp|S4R3P1|HMN13_HUMAN Humanin-like 13 OS=Homo sapiens OX=9606 GN=MTRNR2L13 PE=3 SV=1
>sp|O43869|OR2T1_HUMAN Olfactory receptor 2T1 OS=Homo sapiens OX=9606 GN=OR2T1 PE=3 SV=3
>sp|P0DPF7|TVB63_HUMAN T cell receptor beta variable 6-3 OS=Homo sapiens OX=9606 GN=TRBV6-3 PE=3 SV=1
>sp|P50219|MNX1_HUMAN Motor neuron and pancreas homeobox protein 1 OS=Homo sapiens OX=9606 GN=MNX1 PE=1 SV=3
>sp|Q14390|GGTL2_HUMAN Glutathione hydrolase light chain 2 OS=Homo sapiens OX=9606 GN=GGTLC2 PE=1 SV=4
>sp|Q6DHV7|ADAL_HUMAN Adenosine deaminase-like protein OS=Homo sapiens OX=9606 GN=ADAL PE=1 SV=2
>sp|Q8IX05|CD302_HUMAN CD302 antigen OS=Homo sapiens OX=9606 GN=CD302 PE=1 SV=1
>sp|Q8WWF3|SSMM1_HUMAN Serine-rich single-pass membrane protein 1 OS=Homo sapiens OX=9606 GN=SSMEM1 PE=1 SV=1
>sp|Q9BRX5|PSF3_HUMAN DNA replication complex GINS protein PSF3 OS=Homo sapiens OX=9606 GN=GINS3 PE=1 SV=1
>sp|Q9NVE5|UBP40_HUMAN Ubiquitin carboxyl-terminal hydrolase 40 OS=Homo sapiens OX=9606 GN=USP40 PE=1 SV=3
>sp|Q9Y6Y8|S23IP_HUMAN SEC23-interacting protein OS=Homo sapiens OX=9606 GN=SEC23IP PE=1 SV=1
# FragPipe v19.1ui state cache

# Please edit the following path to point to the correct location.
# In Windows, please replace single '\' with '\\'
database.db-path=D\:\\Databases\\2023-01-25-decoys-contam-Human_SP_Jan2023.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=true
diann.run-specific-protein-q-value=false
diann.unrelated-runs=false
diann.use-predicted-spectra=true
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=true
fragpipe-config.bin-ionquant=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\IonQuant-1.8.10.jar
fragpipe-config.bin-msfragger=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\MSFragger-3.7\\MSFragger-3.7.jar
fragpipe-config.bin-philosopher=C\:\\Users\\protolab\\Downloads\\FragPipe-jre-19.1\\fragpipe\\tools\\philosopher_v4.8.1_windows_amd64\\philosopher.exe
fragpipe-config.bin-python=C\:\\Users\\protolab\\Anaconda3\\python.exe

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=10
ionquant.medium=
ionquant.minfreq=0
ionquant.minions=1
ionquant.minisotopes=1
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=true
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=0/1/2
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=5
msfragger.min_sequence_matches=2
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.00
msfragger.misc.fragger.clear-mz-hi=0
msfragger.misc.fragger.clear-mz-lo=0
msfragger.misc.fragger.digest-mass-hi=5000
msfragger.misc.fragger.digest-mass-lo=500
msfragger.misc.fragger.enzyme-dropdown-1=stricttrypsin
msfragger.misc.fragger.enzyme-dropdown-2=null
msfragger.misc.fragger.precursor-charge-hi=4
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=stricttrypsin
msfragger.search_enzyme_name_2=null
msfragger.search_enzyme_nocut_1=
msfragger.search_enzyme_nocut_2=
msfragger.search_enzyme_sense_1=C
msfragger.search_enzyme_sense_2=C
msfragger.table.fix-mods=0.0,C-Term Peptide,true,-1; 0.0,N-Term Peptide,true,-1; 0.0,C-Term Protein,true,-1; 0.0,N-Term Protein,true,-1; 0.0,G (glycine),true,-1; 0.0,A (alanine),true,-1; 0.0,S (serine),true,-1; 0.0,P (proline),true,-1; 0.0,V (valine),true,-1; 0.0,T (threonine),true,-1; 57.02146,C (cysteine),true,-1; 0.0,L (leucine),true,-1; 0.0,I (isoleucine),true,-1; 0.0,N (asparagine),true,-1; 0.0,D (aspartic acid),true,-1; 0.0,Q (glutamine),true,-1; 0.0,K (lysine),true,-1; 0.0,E (glutamic acid),true,-1; 0.0,M (methionine),true,-1; 0.0,H (histidine),true,-1; 0.0,F (phenylalanine),true,-1; 0.0,R (arginine),true,-1; 0.0,Y (tyrosine),true,-1; 0.0,W (tryptophan),true,-1; 0.0,B ,true,-1; 0.0,J,true,-1; 0.0,O,true,-1; 0.0,U,true,-1; 0.0,X,true,-1; 0.0,Z,true,-1
msfragger.table.var-mods=15.9949,M,true,3; 42.0106,[^,true,1; 79.96633,STY,false,3; -17.0265,nQnC,true,1; -18.0106,nE,true,1; 0.0,site_06,false,1; 0.0,site_07,false,1; 0.0,site_08,false,1; 0.0,site_09,false,1; 0.0,site_10,false,1; 0.0,site_11,false,1; 0.0,site_12,false,1; 0.0,site_13,false,1; 0.0,site_14,false,1; 0.0,site_15,false,1; 0.0,site_16,false,1
msfragger.track_zero_topN=0
msfragger.use_all_mods_in_first_search=false
msfragger.use_topN_peaks=300
msfragger.write_calibrated_mzml=false
msfragger.write_uncalibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
opair.activation1=HCD
opair.activation2=ETD
opair.glyco_db=
opair.max_glycans=4
opair.max_isotope_error=2
opair.min_isotope_error=0
opair.ms1_tol=20
opair.ms2_tol=20
opair.reverse_scan_order=false
opair.run-opair=false
opair.single_scan_type=false
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=false
percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc

percolator.keep-tsv-files=false
percolator.min-prob=0.7
percolator.run-percolator=true
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--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=true
phi-report.remove-contaminants=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.cap_y_ions=
ptmshepherd.decoy_type=1
ptmshepherd.diag_ions=
ptmshepherd.diagextract_mode=false
ptmshepherd.diagmine_diagMinFoldChange=3.0
ptmshepherd.diagmine_diagMinSpecDiff=25
ptmshepherd.diagmine_fragMinFoldChange=3.0
ptmshepherd.diagmine_fragMinPropensity=12.5
ptmshepherd.diagmine_fragMinSpecDiff=25
ptmshepherd.diagmine_minIonsPerSpec=2
ptmshepherd.diagmine_minPeps=25
ptmshepherd.diagmine_mode=false
ptmshepherd.diagmine_pepMinFoldChange=3.0
ptmshepherd.diagmine_pepMinSpecDiff=25
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=2,0.5,0.1
ptmshepherd.prob_dhexY=2,0.5
ptmshepherd.prob_neuacOx=2,0.05,0.2
ptmshepherd.prob_neugcOx=2,0.05,0.2
ptmshepherd.prob_phosphoOx=2,0.05,0.2
ptmshepherd.prob_regY=5,0.5
ptmshepherd.prob_sulfoOx=2,0.05,0.2
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
saintexpress.fragpipe.cmd-opts=
saintexpress.max-replicates=10
saintexpress.run-saint-express=false
saintexpress.virtual-controls=100
speclibgen.easypqp.extras.max_delta_ppm=15
speclibgen.easypqp.extras.max_delta_unimod=0.02
speclibgen.easypqp.extras.rt_lowess_fraction=0
speclibgen.easypqp.fragment.a=false
speclibgen.easypqp.fragment.b=true
speclibgen.easypqp.fragment.c=false
speclibgen.easypqp.fragment.x=false
speclibgen.easypqp.fragment.y=true
speclibgen.easypqp.fragment.z=false
speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM

speclibgen.easypqp.neutral_loss=false
speclibgen.easypqp.rt-cal=noiRT
speclibgen.easypqp.select-file.text=
speclibgen.easypqp.select-im-file.text=
speclibgen.keep-intermediate-files=false
speclibgen.run-speclibgen=true
tab-run.delete_calibrated_mzml=false
tab-run.delete_temp_files=false
tmtintegrator.add_Ref=-1
tmtintegrator.aggregation_method=0
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=TMT-6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.glyco_qval=-1
tmtintegrator.groupby=0
tmtintegrator.log2transformed=true
tmtintegrator.max_pep_prob_thres=0
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=Bridge
tmtintegrator.run-tmtintegrator=false
tmtintegrator.tolerance=20
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=0
tmtintegrator.unique_pep=false
tmtintegrator.use_glycan_composition=false
workdir=D\:\\17915\\Temp
workflow.input.data-type.im-ms=false
workflow.input.data-type.regular-ms=true
workflow.ram=0
workflow.threads=63
CheckCentroid C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" com.dmtavt.fragpipe.util.CheckCentroid D:\17915\FS1_MB_17915_2_29012023.mzML 63 Done in 6225 ms. Process 'CheckCentroid' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --clean --nocheck INFO[15:56:58] Executing Workspace v4.8.1 INFO[15:56:58] Removing workspace WARN[15:56:58] Cannot read file. open .meta\meta.bin: The system cannot find the path specified. INFO[15:56:58] Done Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe workspace --init --nocheck --temp C:\Users\protolab\AppData\Local\Temp\2\e92b1203-0a35-4118-baee-77b879b2cf05 INFO[15:56:59] Executing Workspace v4.8.1 INFO[15:56:59] Creating workspace INFO[15:56:59] Done Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx844G C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\MSFragger-3.7\MSFragger-3.7.jar D:\17915\Temp\fragger.params D:\17915\FS1_MB_17915_2_29012023.mzML MSFragger version MSFragger-3.7 Batmass-IO version 1.28.9 timsdata library version timsdata-2-21-0-4 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. timdTOF .d reading tool. Copyright (c) 2022 by Bruker Daltonics GmbH & Co. KG. All rights reserved. System OS: Windows Server 2019, Architecture: AMD64 Java Info: 11.0.9.1, OpenJDK 64-Bit Server VM, AdoptOpenJDK JVM started with 844 GB memory Checking database... Checking spectral files... D:\17915\FS1_MB_17915_2_29012023.mzML: Scans = 84704 ***FIRST SEARCH**** Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 5 minimum_ratio = 0.0 intensity_transform = 0 activation_types = all remove_precursor_peak = 1 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.10 Da. 344926862 fragments to be searched in 1 slices (5.14 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.63 s

FS1_MB_17915_2_29012023.mzML 10.0 s | deisotoping 5.1 s [progress: 84552/84552 (100%) - 10998 spectra/s] 7.7s | postprocessing 0.8 s ***FIRST SEARCH DONE IN 0.889 MIN**

**MASS CALIBRATION AND PARAMETER OPTIMIZATION ----- --------------- --------------- --------------- --------------- MS1 (Old) MS1 (New) MS2 (Old) MS2 (New)

Run Median MAD Median MAD Median MAD Median MAD

001 Not enough data to perform mass calibration. Using the uncalibrated data.

----- --------------- --------------- --------------- ---------------

Finding the optimal parameters: -------|-------|-------|-------|-------|-------|-------|-------|-------

MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50

-------|-------|-------|-------|-------|-------|-------|-------|-------

Count 4 29 33 29 skip rest

------- ------- ------- ------- ------- ------- ------- Peaks 300_0 200_0 175_0 150_1 125_1 100_1

Count 33 44 51 51 41 skip rest

------- ------- ------- ------- ------- ------- -------

------- -------

Int. 1

------- -------

Count 49

------- -------

------- -------

Rm P. 0

------- -------

Count 51

------- -------

New fragment_mass_tolerance = 10 PPM New use_topN_peaks = 150 New minimum_ratio = 0.010000 New intensity_transform = 0 New remove_precursor_peak = 0 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 3.195 MIN*****

****MAIN SEARCH**** Checking database... Parameters: num_threads = 63 database_name = D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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_mzml = 0 write_uncalibrated_mgf = false isotope_error = 0/1/2 mass_offsets = 0.0 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 = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = 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 = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 500.0 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 = 150 minIonsScoring = 2 min_matched_fragments = 5 minimum_ratio = 0.01 intensity_transform = 0 activation_types = all remove_precursor_peak = 0 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = true variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 variable_mod_04 = -17.0265 nQnC 1 variable_mod_05 = -18.0106 nE 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.05 Da. 368492928 fragments to be searched in 1 slices (5.49 GB total) Operating on slice 1 of 1: Fragment index slice generated in 3.83 s

FS1_MB_17915_2_29012023.mzML 6.1 s | deisotoping 2.0 s [progress: 84552/84552 (100%) - 49101 spectra/s] 1.7s | remapping alternative proteins and postprocessing 3.7 s MAIN SEARCH DONE IN 0.382 MIN

***TOTAL TIME 4.467 MIN**** Process 'MSFragger' finished, exit code: 0 MSFragger move pepxml C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pepXML D:\17915\Temp\FS1_MB_17915_2_29012023.pepXML Process 'MSFragger move pepxml' finished, exit code: 0 MSFragger move pin C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar;/C:/Users/protolab/Downloads/FragPipe-jre-19.1/fragpipe/lib/commons-io-2.11.0.jar com.github.chhh.utils.FileMove --no-err D:\17915\FS1_MB_17915_2_29012023.pin D:\17915\Temp\FS1_MB_17915_2_29012023.pin Process 'MSFragger move pin' finished, exit code: 0 MSBooster [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -Xmx844G -cp "C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\msbooster-1.1.11.jar;C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\batmass-io-1.28.9.jar" Features.MainClass --paramsList D:\17915\Temp\msbooster_params.txt MSBooster v1.1.11 Using 63 threads Generating input file for DIA-NN 2497 unique peptides from 8558 PSMs Writing DIA-NN input file Diann input file generation took 742 milliseconds Input file at D:\17915\Temp\spectraRT.tsv 2497 unique peptides from 8558 PSMs createFull input file generation took 97 milliseconds Input file at D:\17915\Temp\spectraRT_full.tsv Generating DIA-NN predictions C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\diann\1.8.2_beta_8\win\DiaNN.exe --lib D:\17915\Temp\spectraRT.tsv --predict --threads 63 --strip-unknown-mods --mod TMT,229.1629 --predict-n-frag 100 DIA-NN 1.8.2 beta 8 (Data-Independent Acquisition by Neural Networks) Compiled on Sep 15 2022 18:28:57 Current date and time: Mon Feb 6 16:01:33 2023 CPU: AuthenticAMD AMD EPYC 7552 48-Core Processor SIMD instructions: AVX AVX2 FMA SSE4.1 SSE4.2 SSE4a Logical CPU cores: 64 Predicted spectra will be saved in a binary format Thread number set to 63 DIA-NN will use deep learning to predict spectra/RTs/IMs even for peptides carrying modifications which are not recognised by the deep learning predictor. In this scenario, if also generating a spectral library from the DIA data or using the MBR mode, it might or might not be better (depends on the data) to also use the --out-measured-rt option - it's recommended to test it with and without this option Modification TMT with mass delta 229.163 added to the list of recognised modifications for spectral library-based search Deep learning predictor will predict 100 fragments Cannot find a UniMod modification match for TMT: 73.0618 minimal mass discrepancy; using the original modificaiton name

0 files will be processed [0:00] Loading spectral library D:\17915\Temp\spectraRT.tsv [0:00] Finding proteotypic peptides (assuming that the list of UniProt ids provided for each peptide is complete) [0:00] Spectral library loaded: 0 protein isoforms, 0 protein groups and 2497 precursors in 2497 elution groups. [0:00] Encoding peptides for spectra and RTs prediction [0:00] Predicting spectra and IMs [0:05] Predicting RTs [0:07] Decoding predicted spectra and IMs [0:07] Decoding RTs [0:07] Saving the list of predictions to D:\17915\Temp\spectraRT.predicted.bin Finished Done generating DIA-NN predictions Model running took 8217 milliseconds Generating edited pin with following features: [unweightedSpectralEntropy, deltaRTLOESS] Loading predicted spectra Processing FS1_MB_17915_2_29012023_uncalibrated.mzML RT regression using 4 PSMs Edited pin file at D:\17915\Temp\FS1_MB_17915_2_29012023_edited.pin Done in 13476 ms Process 'MSBooster' finished, exit code: 0 Percolator [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Percolator version 3.05.0, Build Date May 18 2020 02:35:01 Copyright (c) 2006-9 University of Washington. All rights reserved. Written by Lukas Käll @.***) in the Department of Genome Sciences at the University of Washington. Issued command: C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\percolator-305\percolator.exe --only-psms --no-terminate --post-processing-tdc --num-threads 63 --results-psms FS1_MB_17915_2_29012023_percolator_target_psms.tsv --decoy-results-psms FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv FS1_MB_17915_2_29012023_edited.pin Started Mon Feb 6 16:01:54 2023 Hyperparameters: selectionFdr=0.01, Cpos=0, Cneg=0, maxNiter=10

Reading tab-delimited input from datafile FS1_MB_17915_2_29012023_edited.pin Features: retentiontime rank abs_ppm isotope_errors log10_evalue hyperscore delta_hyperscore matched_ion_num complementary_ions ion_series weighted_average_abs_fragment_ppm peptide_length ntt nmc charge_1 charge_2 charge_3 charge_4 charge_5 charge_6 charge_7_or_more -17.0265nQnC -18.0106nE 15.9949M unweighted_spectral_entropy delta_RT_loess Found 8558 PSMs Concatenated search input detected and --post-processing-tdc flag set. Applying target-decoy competition on Percolator scores. Train/test set contains 4278 positives and 4280 negatives, size ratio=0.999533 and pi0=1 Selecting Cpos by cross-validation. Selecting Cneg by cross-validation. Split 1: Selected feature 6 as initial direction. Could separate 34 training set positives with q<0.01 in that direction. Split 2: Selected feature 5 as initial direction. Could separate 35 training set positives with q<0.01 in that direction. Split 3: Selected feature 5 as initial direction. Could separate 28 training set positives with q<0.01 in that direction. Found 0 test set positives with q<0.01 in initial direction Reading in data and feature calculation took 0.483 cpu seconds or 1 seconds wall clock time. ---Training with Cpos selected by cross validation, Cneg selected by cross validation, initial_fdr=0.01, fdr=0.01 Iteration 1: Estimated 0 PSMs with q<0.01 Iteration 2: Estimated 59 PSMs with q<0.01 Iteration 3: Estimated 72 PSMs with q<0.01 Iteration 4: Estimated 181 PSMs with q<0.01 Iteration 5: Estimated 188 PSMs with q<0.01 Iteration 6: Estimated 190 PSMs with q<0.01 Iteration 7: Estimated 191 PSMs with q<0.01 Iteration 8: Estimated 194 PSMs with q<0.01 Iteration 9: Estimated 200 PSMs with q<0.01 Iteration 10: Estimated 203 PSMs with q<0.01 Learned normalized SVM weights for the 3 cross-validation splits: Split1 Split2 Split3 FeatureName -4.4913 0.2390 -0.2953 retentiontime 0.0000 0.0000 0.0000 rank 1.0970 1.3365 0.6797 abs_ppm 5.2691 1.9916 2.6877 isotope_errors -2.9522 -3.6102 -3.3298 log10_evalue 0.8751 0.0799 0.7274 hyperscore -0.7880 -0.4236 0.0302 delta_hyperscore -0.8479 -1.4030 -1.6929 matched_ion_num 0.7424 0.1502 -0.4959 complementary_ions 1.7124 1.3828 1.5140 ion_series 0.3132 -0.1346 0.1116 weighted_average_abs_fragment_ppm -0.1715 -0.6610 -0.4867 peptide_length 0.0000 0.0000 0.0000 ntt -0.7360 -1.1251 -1.0992 nmc 0.0000 0.0000 0.0000 charge_1 0.0000 0.0000 0.0000 charge_2 0.0000 0.0000 0.0000 charge_3 0.0000 0.0000 0.0000 charge_4 0.0000 0.0000 0.0000 charge_5 0.0000 0.0000 0.0000 charge_6 0.0000 0.0000 0.0000 charge_7_or_more 0.7435 -0.1741 -0.0229 -17.0265nQnC 0.0273 0.9277 0.5164 -18.0106nE 1.2156 2.1702 1.5007 15.9949M 0.7971 1.4475 0.9711 unweighted_spectral_entropy -6.1394 -3.3990 -3.6452 delta_RT_loess -13.0417 -12.0309 -11.9982 m0 Found 0 test set PSMs with q<0.01. No targets found with q<0.01 Resetting score vector, using default vector. Use --override flag to prevent this. Split 1: Selected feature 6 as initial direction. Could separate 34 training set positives with q<0.01 in that direction. Split 2: Selected feature 5 as initial direction. Could separate 35 training set positives with q<0.01 in that direction. Split 3: Selected feature 5 as initial direction. Could separate 28 training set positives with q<0.01 in that direction. Selected best-scoring PSM per scan+expMass (target-decoy competition): 4278 target PSMs and 4280 decoy PSMs. Calculating q values. Final list yields 0 target PSMs with q<0.01. Calculating posterior error probabilities (PEPs). Processing took 10.89 cpu seconds or 11 seconds wall clock time. Process 'Percolator' finished, exit code: 0 Percolator: Convert to pepxml [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib/* com.dmtavt.fragpipe.tools.percolator.PercolatorOutputToPepXML FS1_MB_17915_2_29012023.pin FS1_MB_17915_2_29012023 FS1_MB_17915_2_29012023_percolator_target_psms.tsv FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv interact-FS1_MB_17915_2_29012023 DDA 0.7 Process 'Percolator: Convert to pepxml' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_target_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 Percolator: Delete temp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\jre\bin\java.exe -cp C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\lib\fragpipe-19.1.jar com.github.chhh.utils.FileDelete D:\17915\Temp\FS1_MB_17915_2_29012023_percolator_decoy_psms.tsv Process 'Percolator: Delete temp' finished, exit code: 0 ProteinProphet [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe proteinprophet --maxppmdiff 2000000 --minprob 0.5 --output combined D:\17915\Temp\filelist_proteinprophet.txt INFO[16:02:09] Executing ProteinProphet v4.8.1 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 D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml... ...read in 0 1+, 0 2+, 18 3+, 0 4+, 0 5+, 0 6+, 0 7+ spectra with min prob 0.5

Initializing 4 peptide weights: 0%...50%...100% Calculating protein lengths and molecular weights from database D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.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 .........:.........:.........:.........:.........:.........:.........:.........:....... Total: 40874 Computing degenerate peptides for 4 proteins: 0%...50%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Computing probabilities for 4 proteins. Loop 1: 0%...100% Loop 2: 0%...100% Calculating sensitivity...and error tables... INFO: mu=6.78873e-09, db_size=45578978 Computing MU for 4 proteins: 0%...50%...100% INFO[16:02:12] Done Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe database --annotate D:\Databases\2023-01-25-decoys-contam-Human_SPJan2023.fasta.fas --prefix rev INFO[16:02:12] Executing Database v4.8.1 INFO[16:02:12] Annotating the database INFO[16:02:23] Done Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windowsamd64\philosopher.exe filter --picked --prot 0.01 --tag rev --pepxml D:\17915\Temp --protxml D:\17915\Temp\combined.prot.xml --razor INFO[16:02:24] Executing Filter v4.8.1 INFO[16:02:24] Processing peptide identification files INFO[16:02:24] Parsing D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml INFO[16:02:24] 1+ Charge profile decoy=0 target=0 INFO[16:02:24] 2+ Charge profile decoy=0 target=0 INFO[16:02:24] 3+ Charge profile decoy=0 target=18 INFO[16:02:24] 4+ Charge profile decoy=0 target=0 INFO[16:02:24] 5+ Charge profile decoy=0 target=0 INFO[16:02:24] 6+ Charge profile decoy=0 target=0 INFO[16:02:24] Database search results ions=4 peptides=4 psms=18 INFO[16:02:24] Converged to 0.00 % FDR with 18 PSMs decoy=0 threshold=0.734154 total=18 INFO[16:02:24] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Protein inference results decoy=0 target=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Proteins decoy=0 threshold=0.8053 total=4 INFO[16:02:24] 2D FDR estimation: Protein mirror image decoy=4 target=4 INFO[16:02:24] Second filtering results ions=4 peptides=4 psms=18 INFO[16:02:24] Converged to 0.00 % FDR with 18 PSMs decoy=0 threshold=0.734154 total=18 INFO[16:02:24] Converged to 0.00 % FDR with 4 Peptides decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Converged to 0.00 % FDR with 4 Ions decoy=0 threshold=0.80613 total=4 INFO[16:02:24] Post processing identifications INFO[16:02:24] Assigning protein identifications to layers INFO[16:02:25] Processing protein inference INFO[16:02:26] Synchronizing PSMs and proteins INFO[16:02:26] Final report numbers after FDR filtering, and post-processing ions=4 peptides=4 proteins=4 psms=18 INFO[16:02:26] Saving INFO[16:02:26] Done Process 'PhilosopherFilter' finished, exit code: 0 PhilosopherReport [Work dir: D:\17915\Temp] C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\philosopher_v4.8.1_windows_amd64\philosopher.exe report INFO[16:02:26] Executing Report v4.8.1 INFO[16:02:26] Creating reports INFO[16:02:26] Done Process 'PhilosopherReport' finished, exit code: 0 SpecLibGen [Work dir: D:\17915\Temp] C:\Users\protolab\Anaconda3\python.exe -u C:\Users\protolab\Downloads\FragPipe-jre-19.1\fragpipe\tools\speclib\gen_con_spec_lib.py D:\Databases\2023-01-25-decoys-contam-Human_SP_Jan2023.fasta.fas D:\17915\Temp unused D:\17915\Temp True unused use_easypqp noiRT;noIM 63 "--max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types [\'b\',\'y\',] " "--rt_lowess_fraction 0.0" delete_intermediate_files D:\17915\Temp\filelist_speclibgen.txt File list provided Spectral library building Commands to execute: 'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' convert --max_delta_unimod 0.02 --max_delta_ppm 15.0 --fragment_types '['"'"'b'"'"','"'"'y'"'"',]' --enable_unannotated --pepxml 'D:\17915\Temp\interact-FS1_MB_17915_2_29012023.pep.xml' --spectra 'D:\17915\FS1_MB_17915_2_29012023.mzML' --exclude-range -1.5,3.5 --psms FS1_MB_17915_2_29012023.psmpkl --peaks FS1_MB_17915_2_29012023.peakpkl

'C:\Users\protolab\Anaconda3\Scripts\easypqp.exe' library --psmtsv 'D:\17915\Temp\psm.tsv' --peptidetsv 'D:\17915\Temp\peptide.tsv' --out easypqp_lib_openswath.tsv --rt_lowess_fraction 0.0 'D:\17915\Temp\filelist_easypqp_library.txt'
easypqp, version 0.1.35
Executing ['C:\\Users\\protolab\\Anaconda3\\Scripts\\easypqp.exe', 'convert', '--max_delta_unimod', '0.02', '--max_delta_ppm', '15.0', '--fragment_types', "['b','y',]", '--enable_unannotated', '--pepxml', 'D:\\17915\\Temp\\interact-FS1_MB_17915_2_29012023.pep.xml', '--spectra', 'D:\\17915\\FS1_MB_17915_2_29012023.mzML', '--exclude-range', '-1.5,3.5', '--psms', 'FS1_MB_17915_2_29012023.psmpkl', '--peaks', 'FS1_MB_17915_2_29012023.peakpkl']
Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
return self._engine.get_loc(casted_key)
File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
return _run_code(code, main_globals, None,
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

exec(code, run_globals)
File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
return self.main(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
rv = self.invoke(ctx)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
rv.append(sub_ctx.command.invoke(sub_ctx))
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
return __callback(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
pepida = pepida.loc[np.isfinite(pepida['irt'])]
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
indexer = self.columns.get_loc(key)
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
raise KeyError(key) from err
KeyError: 'irt'
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 18 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
base_name modified_peptide
0 FS1_MB_17915_2_29012023 4
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 4.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Not enough peptides could be found for alignment.
Using ciRT for alignment
Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3361, in get_loc
return self._engine.get_loc(casted_key)
File "pandas\_libs\index.pyx", line 76, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\index.pyx", line 108, in pandas._libs.index.IndexEngine.get_loc
File "pandas\_libs\hashtable_class_helper.pxi", line 5198, in pandas._libs.hashtable.PyObjectHashTable.get_item
File "pandas\_libs\hashtable_class_helper.pxi", line 5206, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'irt'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 197, in _run_module_as_main
return _run_code(code, main_globals, None,
File "C:\Users\protolab\Anaconda3\lib\runpy.py", line 87, in _run_code

exec(code, run_globals)
File "C:\Users\protolab\Anaconda3\Scripts\easypqp.exe\__main__.py", line 7, in <module>
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1128, in __call__
return self.main(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1053, in main
rv = self.invoke(ctx)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1690, in invoke
rv.append(sub_ctx.command.invoke(sub_ctx))
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 1395, in invoke
return ctx.invoke(self.callback, **ctx.params)
File "C:\Users\protolab\Anaconda3\lib\site-packages\click\core.py", line 754, in invoke
return __callback(*args, **kwargs)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\main.py", line 117, in library
generate(infiles, outfile, psmtsv, peptidetsv, perform_rt_calibration, rt_referencefile, rt_reference_run_path, rt_filter, perform_im_calibration, im_referencefile, im_reference_run_path, im_filter, psm_fdr_threshold, peptide_fdr_threshold, protein_fdr_threshold, rt_lowess_fraction, rt_psm_fdr_threshold, im_lowess_fraction, im_psm_fdr_threshold, pi0_lambda, peptide_plot_path, protein_plot_path, min_peptides, proteotypic, consensus, nofdr)
File "C:\Users\protolab\Anaconda3\lib\site-packages\easypqp\library.py", line 441, in generate
pepida = pepida.loc[np.isfinite(pepida['irt'])]
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\frame.py", line 3458, in __getitem__
indexer = self.columns.get_loc(key)
File "C:\Users\protolab\Anaconda3\lib\site-packages\pandas\core\indexes\base.py", line 3363, in get_loc
raise KeyError(key) from err
KeyError: 'irt'
Info: There are psm.tsv and peptide.tsv. Will ignore --psm_fdr_threshold, --peptide_fdr_threshold, --protein_fdr_threshold, --pi0_lambda, --proteotypic, and --no-proteotypic.
Info: Reading file D:\17915\Temp\FS1_MB_17915_2_29012023.psmpkl.
Info: 18 redundant PSMs identified after filtering with D:\17915\Temp\psm.tsv and D:\17915\Temp\peptide.tsv
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Peptide overlap between run and reference: 0.
Info: easypqp_rt_alignment_FS1_MB_17915_2_29012023; Skipping run because not enough peptides could be found for alignment.
Library not generated, not enough peptides could be found for alignment.

Library not generated, not enough peptides could be found for alignment.

Please try using other options for alignment (e.g. ciRT if used other options)
Process 'SpecLibGen' finished, exit code: 1
Process returned non-zero exit code, stopping
Cancelling 2 remaining tasks

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Nesvilab / FragPipe

Error when running DIA_DIA-Umpire_SpecLib_Quant workflow #993