Peptide Hit Results Processor

The Peptide Hit Results Processor (PHRP) converts search results from various MS/MS identification tools into a series of tab-delimited text files that organize the data in a similar format for each tool. It supports MS-GF+, MaxQuant, MSFragger, MODa, MODPlus, MSAlign, MSPathFinder, TopPIC, and X!Tandem, along with SEQUEST Synopsis/First Hits files.

PHRP will insert modification symbols into the peptide sequences for modified peptides. Parallel files are created containing sequence information, modification details, and protein information. The user can optionally provide a modification definition file that specifies the symbol to use for each modification mass.

Example Data

Example input and output files are in the Data directory:

• MSGFPlus_Example has MS-GF+ results
• XTandem_Example has X!Tandem results

For MS-GF+ results, prior to running PHRP, use the Mzid-To-Tsv-Converter to convert the .mzid file to a tab-delimited .tsv file.

Example Command line

PeptideHitResultsProcRunner.exe /I:Dataset_msgfplus.tsv /M:MSGFPlus_PartTryp_MetOx_20ppmParTol_ModDefs.txt /N:MSGFPlus_PartTryp_MetOx_20ppmParTol.txt /T:Mass_Correction_Tags.txt /L /ProteinMods /F:H_sapiens_UniProt_SPROT_2021-03-07.fasta

Command Line Syntax

PHRP is a console application, and must be run from the Windows command prompt.

• On Linux, use Mono to run the program, for example mono PeptideHitResultsProcRunner.exe

PeptideHitResultsProcRunner.exe
 InputFilePath [/O:OutputDirectoryPath]
 [/BaseName:OutputFileBaseName]
 [/S:[MaxLevel]]
 [/P:ParameterFilePath]
 [/L:[LogFilePath]] [/LogDir:LogDirectoryPath]
 [/T:MassCorrectionTagsFilePath]
 [/M:ModificationDefinitionFilePath]
 [/N:SearchToolParameterFilePath]
 [/F:FastaFilePath]
 [/CreateModSummaryFile:True|False]
 [/ProteinMods] [/ProteinModsIncludeReversed]
 [/UseExistingPepToProteinMapFile]
 [/CreateProteinModsViaPHRP]
 [/IgnorePepToProtMapErrors]
 [/FHT:True|False] [/Syn:True|False]
 [/MaxQScore:50] [/MaxQPEP:0.01]
 [/MSGFPlusSpecEValue:0.0000005] [/MSGFPlusEValue:0.75]
 [/SynProb:0.05] [/SynPValue:0.95]
 [/DB:DatabaseConnectionString]

The input file should be one of the following:

• MS-GF+ results file (_msgfplus.tsv or _msgfdb.tsv or .tsv)
• MSPathFinder results file (_IcTda.txt)
• MaxQuant results files (msms.txt and peptides.txt)
• MSFragger results file (Dataset_psm.tsv or Dataset.tsv)
• MODa results file (_moda.id.txt)
• MODPlus results file (_modp.id.txt)
• MSAlign results file (_MSAlign_ResultTable.txt)
• TopPIC results file (_TopPIC_PrSMs.txt)
• X!Tandem Results file (_xt.xml)
• Legacy tools
  • Inspect results file (_inspect.txt)
  • MSGF-DB results file (_msgfdb.txt)
  • SEQUEST Synopsis File (_syn.txt)
  • SEQUEST First Hits file (_fht.txt)

Optionally use /O to specify the output directory

• If omitted, the output file will be created in the same directory as the input file

Optionally use /BaseName (or /B) to define the base name for the output files

• If omitted, the base name will be auto-defined based on the input file name
• For MaxQuant and MSFragger results, if the input file has multiple datasets, the base name will be either the base name defined by /BaseName or the longest string in common for the dataset names
• This parameter should not be used when the input file spec is a wildcard

Use /S to process all valid files in the input directory and subdirectories

• Include a number after /S (like /S:2) to limit the level of subdirectories to examine

Optionally use /P to supply a parameter file with processing options

• This must be a Key=Value parameter file, for example:
  • PHRP_Options.conf

Use /L to specify that a log file should be created

• Use /L:LogFilePath to specify the name (or full path) for the log file
• Use /LogDir to specify the directory to create the log file (ignored if the LogFilePath is rooted)

Use /T to specify the file containing the mass correction tag info

• This file should be tab delimited, with the first column containing the mass correction tag name and the second column containing the mass (the name cannot contain commas or colons)

Use /M to specify the file containing the modification definitions.

• This file should be tab delimited, with the first column containing the modification symbol, the second column containing the modification mass, plus optionally a third column listing the residues that can be modified with the given mass
• For the third column, use 1 letter residue symbols (no need to separate with commas or spaces)

Use /N to specify the parameter file provided to the search tool

• This is used when processing results from MS-GF+, MSPathFinder, MaxQuant, MSFragger, MODa, MODPlus, MSAlign, TopPIC, and InSpecT
• For MaxQuant, provide either an XML-based parameter file (root element is <MaxQuantParams>) or provide the parameters.txt file created in the txt results directory
  • The XML-based parameter file is preferred, since it is required to allow PHRP to accurately compute monoisotopic masses of peptides identified by MaxQuant

Use /F to specify the path to the FASTA file

• When provided, the order of the proteins in the FASTA file dictates which protein is listed for each peptide in the First Hits file

A modification summary file with extension _ModSummary.txt is created by default

• To disable, use /CreateModSummaryFile:False

Use /ProteinMods to indicate that the _ProteinMods.txt file should be created

• This requires that either an existing _PepToProtMapMTS.txt file exist, or that the FASTA file be defined using /F

Use /ProteinModsIncludeReversed if an existing _ProteinMods.txt file has reversed protein sequences, or if the FASTA file has reversed proteins

• If false, will skip reversed proteins when creating the _ProteinMods.txt file

Use /UseExistingPepToProteinMapFile to use an existing _PepToProtMapMTS.txt file if it exists

Use /CreateProteinModsViaPHRP to indicate that InputFilePath specifies a valid PHRP data file and thus the PHRP data files should not be re-created; only the _ProteinMods.txt file should be created

• This requires that either an existing _PepToProtMapMTS.txt file exist, or that the FASTA file be defined using /F

Use /IgnorePepToProtMapErrors to ignore peptide to protein mapping errors that occur when creating a missing _PepToProtMapMTS.txt file

Use /FHT:True or /FHT:False to control the creation of a first-hits file (_fht.txt)

• The default is /FHT:True

Use /Syn:True or /Syn:False to toggle the creation of a synopsis file (_syn.txt)

• The default is /Syn:True

When processing a MaxQuant results file, use /MaxQScore to customize the Andromeda score threshold used to determine which peptides are written to the synopsis file

• A PSM is stored if its Andromeda score is over the threshold, or if its PEP score is below the threshold
• The default is /MaxQScore:50

When processing a MaxQuant results file, use /MaxQPEP to customize the Posterior Error Probability (PEP) score threshold used to determine which peptides are written to the synopsis file

• The default is /MaxQPEP:0.01

When processing an MS-GF+ results file, use /MSGFPlusSpecEValue and /MSGFPlusEValue to customize the thresholds used to determine which peptides are written to the synopsis file

• Defaults are /MSGFPlusSpecEValue:5E-07 and /MSGFPlusEValue:0.75

When processing a MODPlus or MODa results file, use /SynProb to customize the probability threshold used to determine which peptides are written to the synopsis file

• The default is /SynProb:0.05
• Higher probability values are higher confidence results
  • 0.05 is a very loose filter

When processing a MODPlus or MODa results file, use /SynPValue to customize the p-value threshold used to determine which peptides are written to the synopsis file

• The default is /SynPValue:0.95
• Lower p-values are higher confidence results
  • 0.95 is a very loose filter

When processing MaxQuant results using a computer on the pnl.gov domain, the DMS database is contacted to lookup dataset IDs by dataset name, where dataset name comes from the 'Raw file' column in the msms.txt file

• Optionally use /DB to override the default connection info
• The default is /DB:"host=prismdb2.emsl.pnl.gov;Port=5432;Database=dms;UserId=dmsreader;Password=dms4fun"
• To disable contacting DMS, use /DB:"" or /DB:false

Contacts

Written by Matthew Monroe for the Department of Energy (PNNL, Richland, WA)
E-mail: matthew.monroe@pnnl.gov or proteomics@pnnl.gov
Website: https://github.com/PNNL-Comp-Mass-Spec/ or https://www.pnnl.gov/integrative-omics

License

The Peptide Hit Results Processor is licensed under the 2-Clause BSD License; you may not use this program except in compliance with the License. You may obtain a copy of the License at https://opensource.org/licenses/BSD-2-Clause

Copyright 2018 Battelle Memorial Institute