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Nesvilab / MSFragger

Ultrafast, comprehensive peptide identification for mass spectrometry–based proteomics
https://msfragger.nesvilab.org
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Protein/peptides intensity all 0 #137

Closed Peilou closed 3 years ago

Peilou commented 3 years ago

Hello MSFragger team,

I try to use MSFragger (through FragPipe) to search phosphorylation data from Bruker’s TimsTOF pro. However, all intensity values for proteins and peptides are 0 in the search result. All other results information (like psm) seem okay. I searched directly with raw file .d format without PTM prophet. I have tested IonQuant with protein quant Top N 1, 2, 3. All gave 0 intensity results. I don’t know what other parameters can affect this 0 intensity. Much appreciate your help!

Thank you very much! Pei

fcyu commented 3 years ago

Hi Pei,

Could you send us the log?

Thanks,

Fengchao

Peilou commented 3 years ago

Here is the log. Thank you very much for the quick response.

Pei

On Sat, Mar 6, 2021 at 9:01 PM Fengchao notifications@github.com wrote:

Hi Pei,

Could you send us the log?

Thanks,

Fengchao

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/MSFragger/issues/137#issuecomment-792183539, or unsubscribe https://github.com/notifications/unsubscribe-auth/AHPMXP7DTGDPKKWJ3DSACQDTCLUBVANCNFSM4YXL7HQA .

System OS: Windows 10, Architecture: AMD64 Java Info: 1.8.0_242, OpenJDK 64-Bit Server VM, AdoptOpenJDK

Version info: FragPipe version 14.0 MSFragger version 3.1.1 Philosopher version 3.4.13 (build 1611589727)

LCMS files: Experiment/Group:

14 commands to execute: WorkspaceCleanInit [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --init --nocheck MsFragger [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx107G C:\Users\Proteo-Peaks\Downloads\MSFragger-3.1.1\MSFragger-3.1.1\MSFragger-3.1.1.jar E:\Pei\phos\test2-d-LFQ-tio2-top1\fragger.params I:\ZezongGu\03032021-phos\TiO2-1_2623.d MsFragger move pepxml C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -cp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\lib\fragpipe-14.0.jar com.github.chhh.utils.FileMove I:\ZezongGu\03032021-phos\TiO2-1_2623.pepXML E:\Pei\phos\test2-d-LFQ-tio2-top1\TiO2-1_2623.pepXML PeptideProphet: Workspace init [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1\fragpipe-TiO2-1_2623.pepXML-temp] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --init --nocheck PeptideProphet [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1\fragpipe-TiO2-12623.pepXML-temp] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe peptideprophet --decoyprobs --ppm --accmass --nonparam --expectscore --decoy rev --database C:\Users\Proteo-Peaks\Documents\database\MSFragger_db2\2021-02-26-decoys-reviewed-contam-UP000000589.fas ..\TiO2-1_2623.pepXML PeptideProphet: Delete temp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -cp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\lib\fragpipe-14.0.jar com.github.chhh.utils.FileDelete E:\Pei\phos\test2-d-LFQ-tio2-top1\fragpipe-TiO2-1_2623.pepXML-temp Rewrite pepxml [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -cp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml I:\ZezongGu\03032021-phos\TiO2-1_2623.d ProteinProphet [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe proteinprophet --maxppmdiff 2000000 --output combined E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml PhilosopherDbAnnotate [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe database --annotate C:\Users\Proteo-Peaks\Documents\database\MSFraggerdb2\2021-02-26-decoys-reviewed-contam-UP000000589.fas --prefix rev PhilosopherFilter [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe filter --sequential --razor --prot 0.01 --tag rev_ --pepxml E:\Pei\phos\test2-d-LFQ-tio2-top1 --protxml E:\Pei\phos\test2-d-LFQ-tio2-top1\combined.prot.xml PhilosopherReport [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe report IonQuant [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -Xmx107G -Dlibs.bruker.dir="C:\Users\Proteo-Peaks\Downloads\MSFragger-3.1.1\MSFragger-3.1.1\ext\bruker" -Dlibs.thermo.dir="C:\Users\Proteo-Peaks\Downloads\MSFragger-3.1.1\MSFragger-3.1.1\ext\thermo" -cp "C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\ionquant-1.4.6.jar;C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\batmass-io-1.19.5.jar" ionquant.IonQuant --threads 47 --ionmobility 1 --mbr 0 --proteinquant 1 --requantify 1 --mztol 50 --imtol 0.10 --rttol 0.4 --mbrmincorr 0 --mbrrttol 1 --mbrimtol 0.05 --mbrtoprun 10 --ionfdr 0.01 --proteinfdr 1 --peptidefdr 1 --normalization 1 --minisotopes 2 --writeindex 0 --tp 3 --minfreq 0.5 --minions 1 --minexps 1 --psm psm.tsv --specdir I:\ZezongGu\03032021-phos TiO2-1_2623.pepXML WorkspaceClean [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --clean --nocheck


Execution order:

    Cmd: [START], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [WorkspaceCleanInit], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [MsFragger], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [PeptideProphet], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [ProteinProphet], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [PhilosopherDbAnnotate], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [PhilosopherFilter], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [PhilosopherReport], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [IonQuant], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]
    Cmd: [WorkspaceClean], Work dir: [E:\Pei\phos\test2-d-LFQ-tio2-top1]

# FragPipe v14.0ui state cache

crystalc.run-crystalc=false
database.db-path=C\:\\Users\\Proteo-Peaks\\Documents\\database\\MSFragger_db2\\2021-02-26-decoys-reviewed-contam-UP000000589.fas
database.decoy-tag=rev_
fragpipe-config.bin-msfragger=C\:\\Users\\Proteo-Peaks\\Downloads\\MSFragger-3.1.1\\MSFragger-3.1.1\\MSFragger-3.1.1.jar
fragpipe-config.bin-philosopher=C\:\\Users\\Proteo-Peaks\\Downloads\\FragPipe-jre-14.0\\fragpipe\\tools\\philosopher\\philosopher.exe
fragpipe-config.bin-python=C\:\\Users\\Proteo-Peaks\\anaconda3\\python
freequant.mz-tol=20
freequant.rt-tol=0.4
freequant.run-freequant=false
ionquant.excludemods=
ionquant.heavy=
ionquant.imtol=0.10
ionquant.ionfdr=0.01
ionquant.light=
ionquant.mbr=0
ionquant.mbrimtol=0.05
ionquant.mbrmincorr=0
ionquant.mbrrttol=1
ionquant.mbrtoprun=10
ionquant.medium=
ionquant.minexps=1
ionquant.minfreq=0.5
ionquant.minions=1
ionquant.minisotopes=2
ionquant.mztol=50
ionquant.normalization=1
ionquant.peptidefdr=1
ionquant.proteinfdr=1
ionquant.proteinquant=1
ionquant.requantify=1
ionquant.rttol=0.4
ionquant.run-ionquant=true
ionquant.tp=3
ionquant.writeindex=0
msfragger.Y_type_masses=
msfragger.add_topN_complementary=0
msfragger.allow_multiple_variable_mods_on_residue=false
msfragger.allowed_missed_cleavage=2
msfragger.calibrate_mass=2
msfragger.clip_nTerm_M=true
msfragger.deisotope=1
msfragger.delta_mass_exclude_ranges=(-1.5,3.5)
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=4
msfragger.minimum_peaks=15
msfragger.minimum_ratio=0.01
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=trypsin
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
msfragger.output_max_expect=50
msfragger.output_report_topN=1
msfragger.override_charge=false
msfragger.precursor_mass_lower=-50
msfragger.precursor_mass_mode=selected
msfragger.precursor_mass_units=1
msfragger.precursor_mass_upper=50
msfragger.precursor_true_tolerance=20
msfragger.precursor_true_units=1
msfragger.remove_precursor_peak=0
msfragger.report_alternative_proteins=false
msfragger.restrict_deltamass_to=all
msfragger.run-msfragger=true
msfragger.search_enzyme_butnotafter=P
msfragger.search_enzyme_cutafter=KR
msfragger.search_enzyme_name=trypsin
msfragger.table.fix-mods=0.000000,C-Term Peptide,true,-1; 0.000000,N-Term Peptide,true,-1; 0.000000,C-Term Protein,true,-1; 0.000000,N-Term Protein,true,-1; 0.000000,G (glycine),true,-1; 0.000000,A (alanine),true,-1; 0.000000,S (serine),true,-1; 0.000000,P (proline),true,-1; 0.000000,V (valine),true,-1; 0.000000,T (threonine),true,-1; 57.021460,C (cysteine),true,-1; 0.000000,L (leucine),true,-1; 0.000000,I (isoleucine),true,-1; 0.000000,N (asparagine),true,-1; 0.000000,D (aspartic acid),true,-1; 0.000000,Q (glutamine),true,-1; 0.000000,K (lysine),true,-1; 0.000000,E (glutamic acid),true,-1; 0.000000,M (methionine),true,-1; 0.000000,H (histidine),true,-1; 0.000000,F (phenylalanine),true,-1; 0.000000,R (arginine),true,-1; 0.000000,Y (tyrosine),true,-1; 0.000000,W (tryptophan),true,-1; 0.000000,B ,true,-1; 0.000000,J,true,-1; 0.000000,O,true,-1; 0.000000,U,true,-1; 0.000000,X,true,-1; 0.000000,Z,true,-1
msfragger.table.var-mods=15.994900,M,true,1; 42.010600,[^,true,1; 79.966330,STY,true,1; -17.026500,nQnC,false,1; -18.010600,nE,false,1; 0.000000,site_06,false,1; 0.000000,site_07,false,1; 0.000000,site_08,false,1; 0.000000,site_09,false,1; 0.000000,site_10,false,1; 0.000000,site_11,false,1; 0.000000,site_12,false,1; 0.000000,site_13,false,1; 0.000000,site_14,false,1; 0.000000,site_15,false,1; 0.000000,site_16,false,1
msfragger.track_zero_topN=0
msfragger.use_topN_peaks=150
msfragger.write_calibrated_mgf=false
msfragger.zero_bin_accept_expect=0
msfragger.zero_bin_mult_expect=1
peptide-prophet.cmd-opts=--decoyprobs --ppm --accmass --nonparam --expectscore
peptide-prophet.combine-pepxml=false
peptide-prophet.run-peptide-prophet=true
phi-report.dont-use-prot-proph-file=false
phi-report.filter=--sequential --razor --prot 0.01
phi-report.pep-level-summary=false
phi-report.print-decoys=false
phi-report.run-report=true
protein-prophet.cmd-opts=--maxppmdiff 2000000
protein-prophet.run-protein-prophet=true
ptmprophet.cmdline=--keepold --static --em 1 --nions b --mods STY\:79.966331,M\:15.9949 --minprob 0.5
ptmprophet.run-ptmprophet=false
ptmshepherd.annotation-common=false
ptmshepherd.annotation-custom=false
ptmshepherd.annotation-unimod=true
ptmshepherd.annotation_file=
ptmshepherd.annotation_tol=0.01
ptmshepherd.cap_y_ions=0,203.07937,406.15874,568.21156,730.26438,892.3172,349.137279
ptmshepherd.diag_ions=204.086646,186.076086,168.065526,366.139466,144.0656,138.055,512.197375,292.1026925,274.0921325,657.2349,243.026426,405.079246,485.045576,308.09761
ptmshepherd.glyco_mode=true
ptmshepherd.histo_smoothbins=2
ptmshepherd.iontype_a=false
ptmshepherd.iontype_b=true
ptmshepherd.iontype_c=false
ptmshepherd.iontype_x=false
ptmshepherd.iontype_y=true
ptmshepherd.iontype_z=false
ptmshepherd.localization_background=4
ptmshepherd.output_extended=false
ptmshepherd.peakpicking_mass_units=0
ptmshepherd.peakpicking_promRatio=0.3
ptmshepherd.peakpicking_width=0.002
ptmshepherd.precursor_mass_units=0
ptmshepherd.precursor_tol=0.01
ptmshepherd.remainder_masses=203.07937
ptmshepherd.run-shepherd=false
ptmshepherd.spectra_maxfragcharge=2
ptmshepherd.varmod_masses=Failed_Carbamidomethylation\:-57.021464
quantitation.run-label-free-quant=true
speclibgen.easypqp.extras.rt_lowess_fraction=0.04
speclibgen.easypqp.rt-cal=noiRT
speclibgen.easypqp.select-file.text=
speclibgen.run-speclibgen=false
speclibgen.use-easypqp=false
speclibgen.use-spectrast=true
tmtintegrator.add_Ref=-1
tmtintegrator.allow_overlabel=true
tmtintegrator.allow_unlabeled=true
tmtintegrator.best_psm=true
tmtintegrator.channel_num=6
tmtintegrator.dont-run-fq-lq=false
tmtintegrator.freequant=--ptw 0.4 --tol 10 --isolated
tmtintegrator.groupby=0
tmtintegrator.labelquant=--tol 20 --level 2
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.ref_tag=Bridge
tmtintegrator.run-tmtintegrator=false
tmtintegrator.top3_pep=true
tmtintegrator.unique_gene=0
tmtintegrator.unique_pep=false
workdir=E\:\\Pei\\phos\\test2-d-LFQ-tio2-top1
workflow.input.data-type.im-ms=true
workflow.input.data-type.regular-ms=false
workflow.process-exps-separately=false
workflow.ram=0
workflow.threads=47

WorkspaceCleanInit [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --clean --nocheck Process 'WorkspaceCleanInit' finished, exit code: 0 INFO[20:36:32] Executing Workspace v3.4.13
INFO[20:36:32] Removing workspace
WARN[20:36:32] Cannot read file. open .meta\meta.bin: The system cannot find the path specified. INFO[20:36:32] Done
WorkspaceCleanInit [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --init --nocheck INFO[20:36:32] Executing Workspace v3.4.13
INFO[20:36:32] Creating workspace
INFO[20:36:32] Done
Process 'WorkspaceCleanInit' finished, exit code: 0 MsFragger [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx107G C:\Users\Proteo-Peaks\Downloads\MSFragger-3.1.1\MSFragger-3.1.1\MSFragger-3.1.1.jar E:\Pei\phos\test2-d-LFQ-tio2-top1\fragger.params I:\ZezongGu\03032021-phos\TiO2-1_2623.d MSFragger version MSFragger-3.1.1 Batmass-IO version 1.19.5 timsdata library version timsdata-2-7-0 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. System OS: Windows 10, Architecture: AMD64 Java Info: 1.8.0_242, OpenJDK 64-Bit Server VM, JVM started with 95 GB memory Checking database... Checking I:\ZezongGu\03032021-phos\TiO2-1_2623.d... There is a mzBIN file from I:\ZezongGu\03032021-phos\TiO2-1_2623.d converted with timsdata-2-7-0. Will use this mzBIN file. ***FIRST SEARCH**** Parameters: num_threads = 47 database_name = C:\Users\Proteo-Peaks\Documents\database\MSFragger_db2\2021-02-26-decoys-reviewed-contam-UP000000589.fas decoyprefix = rev precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 20.0 fragment_mass_units = 1 calibrate_mass = 2 write_calibrated_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 = trypsin search_enzyme_cutafter = KR search_enzyme_butnotafter = P num_enzyme_termini = 2 allowed_missed_cleavage = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_file_extension = pepXML output_format = pepXML 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 = 2 deisotope = 1 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 = 4 minimum_ratio = 0.01 intensity_transform = 0 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 variable_mod_01 = 15.994900 M 1 variable_mod_02 = 42.010600 [^ 1 variable_mod_03 = 79.966330 STY 1 add_A_alanine = 0.000000 add_B_user_amino_acid = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_J_user_amino_acid = 0.000000 add_K_lysine = 0.000000 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.000000 # O = pyrrolysine (255.15829 Da) add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_U_user_amino_acid = 0.000000 # U = selenocysteine (150.95363 Da) add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_X_user_amino_acid = 0.000000 add_Y_tyrosine = 0.000000 add_Z_user_amino_acid = 0.000000 Selected fragment index width 0.10 Da. 1262772990 fragments to be searched in 1 slices (18.82 GB total) Operating on slice 1 of 1: Fragment index slice generated in 19.74 s

  1. TiO2-1_2623.mzBIN 9.0 s | deisotoping 3.3 s [progress: 165967/165967 (100%) - 22428 spectra/s] 7.4s | postprocessing 0.7 s ***FIRST SEARCH DONE IN 0.835 MIN**
**MASS CALIBRATION AND PARAMETER OPTIMIZATION ----- --------------- --------------- --------------- --------------- MS1 (Old) MS1 (New) MS2 (Old) MS2 (New)
Run Median MAD Median MAD Median MAD Median MAD
001 2.85 2.74 0.09 2.03 -1.33 6.21 -0.12 5.65
----- --------------- --------------- --------------- ---------------
Finding the optimal parameters: ------- ------- ------- ------- ------- ------- ------- MS2 7 10 15 20 25 30
Count 13673 14916 15332 15677 15182 skip rest
------- ------- ------- ------- ------- ------- -------
------- ------- ------- ------- -------
Peaks 300_0 200_0 150_1 100_1
------- ------- ------- ------- -------
Count 16098 15476 skip rest
------- ------- ------- ------- -------
------- -------
Int. 1
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Count 18206
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Rm P. 1
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Count 18732
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New fragment_mass_tolerance = 20 PPM New use_topN_peaks = 300 New minimum_ratio = 0.000000 New intensity_transform = 1 New remove_precursor_peak = 1 ****MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 4.733 MIN*****

****MAIN SEARCH**** Checking database... Parameters: num_threads = 47 database_name = C:\Users\Proteo-Peaks\Documents\database\MSFragger_db2\2021-02-26-decoys-reviewed-contam-UP000000589.fas decoyprefix = rev precursor_mass_lower = -50.0 precursor_mass_upper = 50.0 precursor_mass_units = 1 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 20.0 fragment_mass_units = 1 calibrate_mass = 2 write_calibrated_mgf = false isotope_error = 0/1/2 mass_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = SELECTED localize_delta_mass = false delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y ion_series_definitions = search_enzyme_name = trypsin search_enzyme_cutafter = KR search_enzyme_butnotafter = P num_enzyme_termini = 2 allowed_missed_cleavage = 2 clip_nTerm_M = true allow_multiple_variable_mods_on_residue = false max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_file_extension = pepXML output_format = pepXML 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 = 2 deisotope = 1 track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 add_topN_complementary = 0 minimum_peaks = 15 use_topN_peaks = 300 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.0 intensity_transform = 1 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 variable_mod_01 = 15.994900 M 1 variable_mod_02 = 42.010600 [^ 1 variable_mod_03 = 79.966330 STY 1 add_A_alanine = 0.000000 add_B_user_amino_acid = 0.000000 add_C_cysteine = 57.021460 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.000000 add_E_glutamic_acid = 0.000000 add_F_phenylalanine = 0.000000 add_G_glycine = 0.000000 add_H_histidine = 0.000000 add_I_isoleucine = 0.000000 add_J_user_amino_acid = 0.000000 add_K_lysine = 0.000000 add_L_leucine = 0.000000 add_M_methionine = 0.000000 add_N_asparagine = 0.000000 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.000000 # O = pyrrolysine (255.15829 Da) add_P_proline = 0.000000 add_Q_glutamine = 0.000000 add_R_arginine = 0.000000 add_S_serine = 0.000000 add_T_threonine = 0.000000 add_U_user_amino_acid = 0.000000 # U = selenocysteine (150.95363 Da) add_V_valine = 0.000000 add_W_tryptophan = 0.000000 add_X_user_amino_acid = 0.000000 add_Y_tyrosine = 0.000000 add_Z_user_amino_acid = 0.000000 Selected fragment index width 0.10 Da. 1262772990 fragments to be searched in 1 slices (18.82 GB total) Operating on slice 1 of 1: Fragment index slice generated in 9.41 s

  1. TiO2-1_2623.mzBIN_calibrated 5.5 s [progress: 165482/165482 (100%) - 13279 spectra/s] 12.5s | postprocessing 11.0 s MAIN SEARCH DONE IN 0.697 MIN

***TOTAL TIME 6.265 MIN**** Process 'MsFragger' finished, exit code: 0 MsFragger move pepxml C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -cp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\lib\fragpipe-14.0.jar com.github.chhh.utils.FileMove I:\ZezongGu\03032021-phos\TiO2-1_2623.pepXML E:\Pei\phos\test2-d-LFQ-tio2-top1\TiO2-1_2623.pepXML Process 'MsFragger move pepxml' finished, exit code: 0 PeptideProphet: Workspace init [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1\fragpipe-TiO2-1_2623.pepXML-temp] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --init --nocheck INFO[20:42:57] Executing Workspace v3.4.13
INFO[20:42:57] Creating workspace
INFO[20:42:57] Done
Process 'PeptideProphet: Workspace init' finished, exit code: 0 PeptideProphet [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1\fragpipe-TiO2-12623.pepXML-temp] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe peptideprophet --decoyprobs --ppm --accmass --nonparam --expectscore --decoy rev --database C:\Users\Proteo-Peaks\Documents\database\MSFragger_db2\2021-02-26-decoys-reviewed-contam-UP000000589.fas ..\TiO2-1_2623.pepXML INFO[20:42:57] Executing PeptideProphet v3.4.13
Unknown file type. No file loaded.E:\Pei\phos\test2-d-LFQ-tio2-top1/TiO2-1_2623.d WARNING: cannot open data file E:\Pei\phos\test2-d-LFQ-tio2-top1/TiO2-1_2623.d in msms_run_summary tag... unrecognized extension .d, trying .mzML ... WARNING: CANNOT correct data file E:\Pei\phos\test2-d-LFQ-tio2-top1/TiO2-1_2623.mzML in msms_run_summary tag... Unknown file type. No file loaded.E:\Pei\phos\test2-d-LFQ-tio2-top1/TiO2-1_2623.d WARNING: cannot open data file E:\Pei\phos\test2-d-LFQ-tio2-top1/TiO2-1_2623.d in msms_run_summary tag... unrecognized extension .d, trying .mzML ... WARNING: CANNOT correct data file E:\Pei\phos\test2-d-LFQ-tio2-top1/TiO2-1_2623.mzML in msms_run_summary tag... file 1: E:\Pei\phos\test2-d-LFQ-tio2-top1\TiO2-1_2623.pepXML processed altogether 132310 results INFO: Results written to file: E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml

using Accurate Mass Bins using PPM mass difference Using Decoy Label "rev_". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling) adding ACCMASS mixture distribution using search_offsets in ACCMASS mixture distr: 0 init with X! Tandem trypsin PeptideProphet (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) AKeller@ISB read in 528 1+, 65707 2+, 37846 3+, 20617 4+, 7612 5+, 0 6+, and 0 7+ spectra. Found 52777 Decoys, and 79533 Non-Decoys MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN

INFO: Processing standard MixtureModel ... Initialising statistical models ... Iterations: .........10.........20....... WARNING: Mixture model quality test failed for charge (6+). WARNING: Mixture model quality test failed for charge (7+). model complete after 28 iterations INFO[20:48:05] Done
Process 'PeptideProphet' finished, exit code: 0 PeptideProphet: Delete temp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -cp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\lib\fragpipe-14.0.jar com.github.chhh.utils.FileDelete E:\Pei\phos\test2-d-LFQ-tio2-top1\fragpipe-TiO2-1_2623.pepXML-temp Process 'PeptideProphet: Delete temp' finished, exit code: 0 Rewrite pepxml [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -cp C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\lib/* com.dmtavt.fragpipe.util.RewritePepxml E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml I:\ZezongGu\03032021-phos\TiO2-1_2623.d Fixing pepxml: E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml Writing output to: E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml1681247921465374779.temp-rewrite Deleting file: E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml Moving rewritten file to original location: [E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml1681247921465374779.temp-rewrite] -> [E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml] Process 'Rewrite pepxml' finished, exit code: 0 ProteinProphet [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe proteinprophet --maxppmdiff 2000000 --output combined E:\Pei\phos\test2-d-LFQ-tio2-top1\interact-TiO2-1_2623.pep.xml INFO[20:48:08] Executing ProteinProphet v3.4.13
ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) (no FPKM) (using degen pep info) Reading in E:/Pei/phos/test2-d-LFQ-tio2-top1/interact-TiO2-1_2623.pep.xml... ...read in 85 1+, 14893 2+, 6130 3+, 2625 4+, 762 5+, 0 6+, 0 7+ spectra with min prob 0.05

Initializing 15279 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating protein lengths and molecular weights from database c:/Users/Proteo-Peaks/Documents/database/MSFragger_db2/2021-02-26-decoys-reviewed-contam-UP000000589.fas .........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........14000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........15000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........16000 .......WARNING: Found the following zero-mass residues in protein entry rev_sp|Q9JLT4|TRXR2_MOUSE : U GUCGTVTPELGSRKSIHLKVVEESCTPHIGVTQMVQAYSAGCKIGLAFGQTVEGANPGLFHLGLVLQPPERMCVMKIYCQSADRDAVTFELPKYYAHYVEVHEQGHLAVAEEESLGVCGYELPTFVTTPVNSYDMLTSSKGFLRQALLKGAKIATPTLEPRGEAVDGIAYIHPVSTAEQADVIIKQNKPNTSIGAKELNLTRTEPVRGIAWLVTDFTGTDEKGSAHDEWTVQLQNTPLKKIHSPVCGKLFQTGHSEMHETVLSSMQQDFGRLPISRMMVTTDLGIGTLFGACELAVYSAGVVLTKGPSEKLWFIDDSTIGYELAGKVQTPYRPRGGTAIVIHEASLLTAKGGKDVGRVTHEDVFSAKINFYKVKRDQLQVRHGWNLSKVHNQVAEAMTKWNHQVPQAVEWGYHHADRIMGGLLAAQHMLKKPICGVNVCTGGLGWKTGRPSPEVYDAVAVKKGLQAAEKACALGGSGGGIVLLDFSQQGGAASAAGRTGRTLARTRPRFRRSPGRLAAVMAAVM ..:.........:.........:.........:.........:.........:.........:.........:.........:.........17000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........18000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........19000 .........:.........:.........:.........:.........:.......WARNING: Found the following zero-mass residues in protein entry sp|P11352|GPX1_MOUSE : U MCAARLSAAAQSTVYAFSARPLTGGEPVSLGSLRGKVLLIENVASLUGTTIRDYTEMNDLQKRLGPRGLVVLGFPCNQFGHQENGKNEEILNSLKYVRPGGGFEPNFTLFEKCEVNGEKAHPLFTFLRNALPTPSDDPTALMTDPKYIIWSPVCRNDIAWNFEKFLVGPDGVPVRRYSRRFRTIDIEPDIETLLSQQSGNS ..:.........:.........:.........:.........20000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........21000 .........:.........:.........:.........:.WARNING: Found the following zero-mass residues in protein entry sp|P62342|SELT_MOUSE : U MRLLLLLLVAASAVVRSEASANLGGVPSKRLKMQYATGPLLKFQICVSUGYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASFLSVFKLVLIGLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMVFFLSNMIENQCMSTGAFEITLNDVPVWSKLESGHLPSMQQLVQILDNEMKLNVHMDSIPHHRS ........:.........:.........:.........:.......WARNING: Found the following zero-mass residues in protein entry sp|P97364|SPS2_MOUSE : U MAEAAAAGASGETMAALVAAEGSLGPAGWSAGRSFSNYRPFEPQTLGFSPSWRLTSFSGMKGUGCKVPQETLLKLLEGLTRPALQPPLTSGLVGGQEETVQEGGLSTRPGPGSAFPSLSIGMDSCVIPLRHGGLSLVQTTDFFYPLVEDPYMMGRIACANVLSDLYAMGITECDNMLMLLSVSQSMSEKEREKVTPLMIKGFRDAAEEGGTAVTGGQTVVNPWIIIGGVATVVCQQNEFIMPDSAVVGDVLVLTKPLGTQVAANAHQWLDNPEKWNKIKMVVSREEVELAYQEAMFNMATLNRTAAGLMHTFNAHAATDITGFGILGHSQNLAKQQKNEVSFVIHNLPIIAKMAAISKASGRFGLLQGTSAETSGGLLICLPREQAARFCSEIKSSKYGEGHQAWIVGIVEKGNRTARIIDKPRVIEVLPRGASAAAAAAPDNSNAASEPSS ..:.........22000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........23000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........24000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........25000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........26000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........27000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........28000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........29000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........30000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........31000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........32000 .........:.........:.........:.........:.........:.........:.........:.....WARNING: Found the following zero-mass residues in protein entry sp|Q9ERR7|SEP15_MOUSE : U MAAGQGGWLRPALGLRLLLATAFQAVSALGAEFASEACRELGFSSNLLCSSCDLLGQFNLLPLDPVCRGCCQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSEKLERI ....:.......WARNING: Found the following zero-mass residues in protein entry sp|Q9JHC0|GPX2_MOUSE : U MAYIAKSFYDLSAIGLDGEKIDFNTFRGRAVLIENVASLUGTTTRDYNQLNELQCRFPRRLVVLGFPCNQFGHQENCQNEEILNSLKYVRPGGGYQPTFSLTQKCDVNGQNEHPVFAYLKDKLPYPYDDPFSLMTDPKLIIWSPVRRSDVSWNFEKFLIGPEGEPFRRYSRSFQTINIEPDIKRLLKVAI ..:.........33000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........34000 .........:.........:.........:.... Total: 34348 Computing degenerate peptides for 5304 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Computing probabilities for 5383 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 5383 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 5383 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 5383 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing 5187 protein groups: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating sensitivity...and error tables... INFO: mu=6.15637e-05, db_size=38777775 Computing MU for 5383 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100%

Finished. INFO[20:48:29] Done
Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe database --annotate C:\Users\Proteo-Peaks\Documents\database\MSFraggerdb2\2021-02-26-decoys-reviewed-contam-UP000000589.fas --prefix rev INFO[20:48:29] Executing Database v3.4.13
INFO[20:48:29] Processing database
Process 'PhilosopherDbAnnotate' finished, exit code: 0 INFO[20:48:32] Done
PhilosopherFilter [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe filter --sequential --razor --prot 0.01 --tag rev_ --pepxml E:\Pei\phos\test2-d-LFQ-tio2-top1 --protxml E:\Pei\phos\test2-d-LFQ-tio2-top1\combined.prot.xml INFO[20:48:32] Executing Filter v3.4.13
INFO[20:48:32] Processing peptide identification files
INFO[20:48:36] 1+ Charge profile decoy=14 target=71 INFO[20:48:36] 2+ Charge profile decoy=808 target=14087 INFO[20:48:36] 3+ Charge profile decoy=220 target=5911 INFO[20:48:36] 4+ Charge profile decoy=81 target=2544 INFO[20:48:36] 5+ Charge profile decoy=29 target=733 INFO[20:48:36] 6+ Charge profile decoy=0 target=0 INFO[20:48:36] Database search results ions=14855 peptides=11657 psms=24498 INFO[20:48:36] Converged to 1.00 % FDR with 20549 PSMs decoy=207 threshold=0.6196 total=20756 INFO[20:48:36] Converged to 1.00 % FDR with 8786 Peptides decoy=88 threshold=0.8452 total=8874 INFO[20:48:36] Converged to 1.00 % FDR with 11778 Ions decoy=118 threshold=0.7761 total=11896 INFO[20:48:38] Protein inference results decoy=1035 target=4152 INFO[20:48:38] Converged to 1.02 % FDR with 3048 Proteins decoy=31 threshold=0.9632 total=3079 INFO[20:48:39] Applying sequential FDR estimation ions=11824 peptides=8894 psms=20296 INFO[20:48:39] Converged to 0.22 % FDR with 20250 PSMs decoy=46 threshold=0.6207 total=20296 INFO[20:48:39] Converged to 0.35 % FDR with 8863 Peptides decoy=31 threshold=0.6207 total=8894 INFO[20:48:39] Converged to 0.27 % FDR with 11792 Ions decoy=32 threshold=0.6207 total=11824 INFO[20:48:40] Post processing identifications
INFO[20:48:41] Mapping modifications
INFO[20:48:47] Processing protein inference
INFO[20:48:54] Assigning protein identifications to layers
INFO[20:48:54] Updating razor PSM assignment to proteins
INFO[20:48:55] Calculating spectral counts
INFO[20:48:55] Saving
INFO[20:48:57] Done
Process 'PhilosopherFilter' finished, exit code: 0 PhilosopherReport [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe report INFO[20:48:58] Executing Report v3.4.13
INFO[20:49:01] Creating reports
Process 'PhilosopherReport' finished, exit code: 0 INFO[20:49:02] Done
IonQuant [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\jre\bin\java.exe -Xmx107G -Dlibs.bruker.dir="C:\Users\Proteo-Peaks\Downloads\MSFragger-3.1.1\MSFragger-3.1.1\ext\bruker" -Dlibs.thermo.dir="C:\Users\Proteo-Peaks\Downloads\MSFragger-3.1.1\MSFragger-3.1.1\ext\thermo" -cp "C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\ionquant-1.4.6.jar;C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\batmass-io-1.19.5.jar" ionquant.IonQuant --threads 47 --ionmobility 1 --mbr 0 --proteinquant 1 --requantify 1 --mztol 50 --imtol 0.10 --rttol 0.4 --mbrmincorr 0 --mbrrttol 1 --mbrimtol 0.05 --mbrtoprun 10 --ionfdr 0.01 --proteinfdr 1 --peptidefdr 1 --normalization 1 --minisotopes 2 --writeindex 0 --tp 3 --minfreq 0.5 --minions 1 --minexps 1 --psm psm.tsv --specdir I:\ZezongGu\03032021-phos TiO2-1_2623.pepXML IonQuant version IonQuant-1.4.6 Batmass-IO version 1.19.5 timsdata library version timsdata-2-7-0 (c) University of Michigan System OS: Windows 10, Architecture: AMD64 Java Info: 1.8.0_242, OpenJDK 64-Bit Server VM, JVM started with 95 GB memory 2021-03-06 20:49:04 [INFO] - Parameters: 2021-03-06 20:49:04 [INFO] - threads = 47 2021-03-06 20:49:04 [INFO] - mztol = 50.0 2021-03-06 20:49:04 [INFO] - rttol = 0.4 2021-03-06 20:49:04 [INFO] - imtol = 0.1 2021-03-06 20:49:04 [INFO] - seedmz = 0 2021-03-06 20:49:04 [INFO] - minisotopes = 2 2021-03-06 20:49:04 [INFO] - psm = E:\Pei\phos\test2-d-LFQ-tio2-top1\psm.tsv 2021-03-06 20:49:04 [INFO] - multidir = null 2021-03-06 20:49:04 [INFO] - excludemods = 2021-03-06 20:49:04 [INFO] - minions = 1 2021-03-06 20:49:04 [INFO] - proteinquant = 1 2021-03-06 20:49:04 [INFO] - normalization = 1 2021-03-06 20:49:04 [INFO] - minexps = 1 2021-03-06 20:49:04 [INFO] - minfreq = 0.5 2021-03-06 20:49:04 [INFO] - tp = 3 2021-03-06 20:49:04 [INFO] - mbr = 0 2021-03-06 20:49:04 [INFO] - mbrrttol = 1.0 2021-03-06 20:49:04 [INFO] - mbrimtol = 0.05 2021-03-06 20:49:04 [INFO] - mbrtoprun = 10 2021-03-06 20:49:04 [INFO] - mbrmincorr = 0.0 2021-03-06 20:49:04 [INFO] - ionmobility = 1 2021-03-06 20:49:04 [INFO] - ionfdr = 0.01 2021-03-06 20:49:04 [INFO] - peptidefdr = 1.0 2021-03-06 20:49:04 [INFO] - proteinfdr = 1.0 2021-03-06 20:49:04 [INFO] - light = 2021-03-06 20:49:04 [INFO] - medium = 2021-03-06 20:49:04 [INFO] - heavy = 2021-03-06 20:49:04 [INFO] - requantify = 0 2021-03-06 20:49:04 [INFO] - writeindex = 0 2021-03-06 20:49:04 [INFO] - specdir = I:\ZezongGu\03032021-phos 2021-03-06 20:49:04 [INFO] - E:\Pei\phos\test2-d-LFQ-tio2-top1\TiO2-1_2623.pepXML 2021-03-06 20:49:04 [INFO] - Checking I:\ZezongGu\03032021-phos\TiO2-1_2623.mzML... 2021-03-06 20:49:05 [INFO] - Loading I:\ZezongGu\03032021-phos\TiO2-1_2623.mzML... 2021-03-06 20:49:09 [INFO] - Building index... 2021-03-06 20:49:09 [INFO] - Loading E:\Pei\phos\test2-d-LFQ-tio2-top1\TiO2-1_2623.pepXML... 2021-03-06 20:49:15 [INFO] - Use each MS2 scan's calculated MZ in peak tracing. 2021-03-06 20:49:15 [INFO] - Quantifying... 2021-03-06 20:49:15 [INFO] - Updating Philosopher's tables... 2021-03-06 20:49:16 [WARNING] - Peptide SPGEAKSPGEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPGEAKSPSEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPGEAKSPSEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPGEAKSPSEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPGEAKSPAEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPSEAKSPAEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEAKSPAEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEAKSPAEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEAKSPAEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEAKSPAEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPGEAKSPAEPK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPATVKSPGEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEVKSPGEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEVKSPGEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEVKSPGEAK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:16 [WARNING] - Peptide SPAEAKSPAEVK is shown to be not unique but does not have mapped proteins. Treating it as a unique peptide. 2021-03-06 20:49:17 [INFO] - Done! Process 'IonQuant' finished, exit code: 0 WorkspaceClean [Work dir: E:\Pei\phos\test2-d-LFQ-tio2-top1] C:\Users\Proteo-Peaks\Downloads\FragPipe-jre-14.0\fragpipe\tools\philosopher\philosopher.exe workspace --clean --nocheck INFO[20:49:17] Executing Workspace v3.4.13
INFO[20:49:17] Removing workspace
INFO[20:49:17] Done
Process 'WorkspaceClean' finished, exit code: 0

Please cite: (Regular searches) MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. Nat Methods 14:513 (2017) (Open search) Identification of modified peptides using localization-aware open search. Nat Commun. 11:4065 (2020) (Open search) Crystal-C: A Computational Tool for Refinement of Open Search Results. J. Proteome Res. 19.6:2511 (2020)

fcyu commented 3 years ago

Your log looks good. Could you upload all of the files generated by FragPipe, fasta, and .d to https://umich.app.box.com/f/2cff9c63061b404fa3df59f83eeca712 ?

Thanks,

Fengchao

Peilou commented 3 years ago

Has been submitted. Thank you!

Pei

On Sat, Mar 6, 2021 at 9:20 PM Fengchao notifications@github.com wrote:

Your log looks good. Could you upload all of the files generated by FragPipe, fasta, and .d to https://umich.app.box.com/f/2cff9c63061b404fa3df59f83eeca712 ?

Thanks,

Fengchao

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/MSFragger/issues/137#issuecomment-792188466, or unsubscribe https://github.com/notifications/unsubscribe-auth/AHPMXP6YC4KMY6S6KKIZKVDTCLWJRANCNFSM4YXL7HQA .

fcyu commented 3 years ago

Hi Pei,

Does your I:\ZezongGu\03032021-phos folder have both .d and .mzML of the same run? If so, can you remove the .mzML file and try again?

Thanks,

Fengchao

Peilou commented 3 years ago

Yes, I have both .d and .mzML in the same folder. After removing .mzML, I did get intensity values. Thank you very much!! Because this is phosphorylation data, I wanted to use PTM Prophet. After searching with .mzML, the intensity value still 0. Could I get intensity with .mzML file? Thanks very much again.

Pei

On Sat, Mar 6, 2021 at 9:39 PM Fengchao notifications@github.com wrote:

Hi Pei,

Does your I:\ZezongGu\03032021-phos folder have both .d and .mzML of the same run? If so, can you remove the .mzML file and try again?

Thanks,

Fengchao

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/MSFragger/issues/137#issuecomment-792192461, or unsubscribe https://github.com/notifications/unsubscribe-auth/AHPMXP32Q5MSTSJ5NI4RN5LTCLYQFANCNFSM4YXL7HQA .

fcyu commented 3 years ago

Hi Pei,

Could you tell me how did you convert .d to .mzML? A screenshot of msconvert would be enough.

Thanks,

Fengchao

Peilou commented 3 years ago

Here is the MSconvert screenshot: [image: image.png]

Thanks Pei

On Sun, Mar 7, 2021 at 9:10 AM Fengchao notifications@github.com wrote:

Hi Pei,

Could you tell me how did you convert .d to .mzML? A screenshot of msconvert would be enough.

Thanks,

Fengchao

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub https://github.com/Nesvilab/MSFragger/issues/137#issuecomment-792294119, or unsubscribe https://github.com/notifications/unsubscribe-auth/AHPMXPYSBNR74ZFEKNLZV5TTCOJOZANCNFSM4YXL7HQA .

fcyu commented 3 years ago

It looks like the image was not successfully sent. Could you send it to yufe@umich.edu?

Thanks,

Fengchao

fcyu commented 3 years ago

Thanks Pei, I've received your figure. I put it here so that we can continue the discussion.

I think the cause it the count 150 most-intense. Top 150 peaks are enough for identification with MS2, but not enough for MS1 quantification. If you are interested in, you can convert the file again without that filter and try again. You should be able to see non-zero intensities.

image

best,

Fengchao

Peilou commented 3 years ago

Hi Fengchao, I removed the count filter 150, and even the scan summing filter, neither gave me intensity.

Thanks, Pei

fcyu commented 3 years ago

Hmm, I will investigate.

Best,

Fengchao

fcyu commented 3 years ago

Hi Pei,

I converted your .d to mzML using the following setting, ran IonQuant with your result folder, and got non-zero intensities from psm.tsv, ion.tsv, peptide.tsv, and protein.tsv. image

I uploaded my result folder (FragPipe-results_yufe.zip) to https://umich.box.com/s/b34hpm7hzrlgdkzg7h7ilzog4zz8eajd for you to troubshooting. The mzML file (22 GB) is too big to upload.

Best,

Fengchao

Peilou commented 3 years ago

Hi Fengchao,

Thank you very much for your help. Much appreciate it! I have converted the .d file to .mzML according to the method you posted, in which the only difference is unchecking "zlib compression". After running MSFragger using the converted .mzML file, and with PTMprophet, ionQuant, the intensity values in my results are still 0. I checked your result fragger.params and log_2021-03-07_20-45-04.txt. I generally used the same parameters. The difference I noticed is my MSFragger is version 14, but yours is 15. Will it be the problem? Thanks again for your kind help. Pei

fcyu commented 3 years ago

Hi Pei,

Version 14 or 15 should not matter. I have no clue now. If you want, I can send you the version I was using.

Best,

Fengchao

Peilou commented 3 years ago

Yes, I would like to. Thank you very much! Pei

fcyu commented 3 years ago

I put it to the folder https://umich.app.box.com/folder/132997293780?s=b34hpm7hzrlgdkzg7h7ilzog4zz8eajd.

Best,

Fengchao

Peilou commented 3 years ago

Thank you very much! Using v15, I got the intensity values!

Pei