The Informed Proteomics project includes algorithms for proteomic mass spectrometry data analysis. Although the back-end data access and some of the scoring routines are general purpose, this repository is currently maintained for top down MS/MS datasets.
The latest versions of the Informed Proteomics tools are available on the AppVeyor CI server
MSPathFinder finds peptides in top-down LC-MS/MS datasets. Similar to database search engines for bottom-up, it takes a fasta file, a spectrum file, and a list of modifications as an input and reports proteoform spectrum matches (PsSMs) and their scores. These results are output in a tab-separated format and in a MzIdentML file.
- Run PbfGen.exe to convert the instrument file or .mzML file to an optimized binary file
- Creates a .pbf file This file contains spectra information and full chromatograms for MS1 and MSn data, allowing fast access to extracted ion chromatograms during the search.
- This step usually only needs to be performed once for a dataset, and by default will do nothing if it has previously been run on the dataset and can find the file.
- ProMex and MSPathFinderT will perform this step automatically if they are given a spectrum file that is not a .pbf file.
- Run ProMex on the .Pbf file to deisotope the data, including determine charge states
- Creates a .ms1ft file
- This file can be reused for multiple searches, as long as none of its parameters change
- MSPathFinderT will perform this step automatically if not provided with a path to a feature file, using the respective parameters from the MSPathFinderT parameters.
- Run MSPathfinderT with the .Pbf (or .raw) file and a fasta file to search for proteins
- Creates _IcTda.tsv files and .mzid
- Can also be run with the spectrum source file directly, with PbfGen and ProMex run as part of the process.
Example command lines:
PbfGen.exe -s MyDataset.raw
ProMex.exe -i MyDataset.pbf -minCharge 2 -maxCharge 60 -minMass 3000 -maxMass 50000 -score n -csv n -maxThreads 0
MSPathFinderT.exe -s MyDataset.pbf -feature MyDataset.ms1ft -d C:\FASTA\ProteinList.fasta -o C:\WorkFolder -t 10 -f 10 -m 1 -tda 1 -minLength 21 -maxLength 300 -minCharge 2 -maxCharge 30 -minFragCharge 1 -maxFragCharge 15 -minMass 3000 -maxMass 50000 -mod MSPathFinder_Mods.txt
Results viewer / GUI
For viewing search results, you might want to consider LCMS-Spectator. It can also function as a GUI for running ProMex and MSPathFinder.
ProMex version 1.0.6232 (January 23, 2017) Usage: ProMex.exe [-i InputFolder or InputFile] [-o OutFolder (default : InputFolder)] [-minCharge MinCharge] (minimum charge state, default: 1) [-maxCharge MaxCharge] (maximum charge state, default: 60) [-minMass MinMassInDa] (minimum mass in Da, default: 2000.0) [-maxMass MaxMassInDa] (maximum mass in Da, default: 50000.0) [-featureMap y or n (default: y)] [-score y or n (default: n)] [-maxThreads 0 (default: 0 (automatic set))] Syntax to create a PNG of the features in an existing ms1ft file (requires both a .pbf file and a .ms1ft file) ProMex.exe [-i PbfFile] [-o OutFolder (default : InputFolder)] [-minMass MinMassInDa] (minimum mass in Da, default: 2000.0) [-maxMass MaxMassInDa] (maximum mass in Da, default: 50000.0) [-ms1ft FeaturesFilePath (use a period to infer the name from the pbf file)]
PbfGen version 1.0.6232 (January 23, 2017) Usage: PbfGen.exe -s RawFilePath (*.raw or directory) [-o OutputDir]
MSPathFinderT version 1.0.7569 (September 21, 2020) Usage: MSPathFinderT.exe -s, arg#1 Required. Spectrum File (*.raw) -d, -database Required. Database File (*.fasta or *.fa or *.faa) -o, -outputDir Output Directory -ic Search Mode (Default: SingleInternalCleavage (or 1)) Possible values are: 0 or 'NoInternalCleavage': No Internal Cleavage 1 or 'SingleInternalCleavage': Single Internal Cleavage 2 or 'MultipleInternalCleavages': Multiple Internal Cleavages -tagSearch Include Tag-based Search (use true or false; or use '0' for false or '1' for true) (Default: True) -n Number of results to report for each mass spectrum (Default: 1) -IncludeDecoys Include decoy results in the _IcTda.tsv file (Default: False) -mod Path to modification file that defines static and dynamic modifications. Modifications can alternatively be defined in a parameter file, as specified by /ParamFile or -ParamFile Modifications defined using the -mod switch take precedence over modifications defined in a parameter file (Default: empty string, meaning no modifications) -tda Database search mode: 0: don't search decoy database, 1: search shuffled decoy database (Default: 0, Min: -1, Max: 1) -t, -precursorTol Precursor Tolerance (in PPM) (Default: 10) -f, -fragmentTol Fragment Ion Tolerance (in PPM) (Default: 10) -minLength Minimum Sequence Length (Default: 21, Min: 0) -maxLength Maximum Sequence Length (Default: 500, Min: 0) -minCharge Minimum precursor ion charge (Default: 2, Min: 1) -maxCharge Maximum precursor ion charge (Default: 50, Min: 1) -minFragCharge Minimum fragment ion charge (Default: 1, Min: 1) -maxFragCharge Maximum fragment ion charge (Default: 20, Min: 1) -minMass Minimum sequence mass in Da (Default: 3000) -maxMass Maximum sequence mass in Da (Default: 50000) -feature *.ms1ft, *_isos.csv, or *.msalign (Default: Run ProMex) -threads Maximum number of threads, or 0 to set automatically (Default: 0, Min: 0) -act Activation Method (Default: Unknown (or 6)) Possible values are: 0 or 'CID' 1 or 'ETD' 2 or 'HCD' 3 or 'ECD' 4 or 'PQD' 5 or 'UVPD' 6 or 'Unknown' -scansFile Text file with MS2 scans to process -flip If specified, FLIP scoring code will be used (supports UVPD spectra) (Default: False) -ParamFile Path to a file containing program parameters. Additional arguments on the command line can supplement or override the arguments in the param file. Lines starting with '#' or ';' will be treated as comments; blank lines are ignored. Lines that start with text that does not match a parameter will also be ignored. -CreateParamFile Create an example parameter file. Can supply a path; if path is not supplied, the example parameter file content will output to the console. NOTE: arg#1, arg#2, etc. refer to positional arguments, used like "AppName.exe [arg#1] [arg#2] [other args]".
Enabling tag-based searching with
-tagSearch 1 can give 5% to 10% more matches, but can increase the runtime by 30% to 50%.
Supported file formats
These programs, when used with no other software installed, only support the use of centroid mzML files as spectrum input.
Versions prior to February 1, 2019: If Thermo Finnigan MSFileReader is installed, it also supports reading from Thermo Finnigan .raw files (Download here, requires registration to download).
Versions after February 1, 2019: If running as a 64-bit program, reading from Thermo Finnigan .raw files is supported via the included RawFileReader dlls (no additional software install required).
Several other formats are supported if an appropriate version of ProteoWizard is installed (Download here, make sure the version downloaded matches system architecture)
MSPathFinder Parameter Files
See the Example_Files folder for sample parameter files
- Example command for invoking MSPathFinder with a parameter file:
MSPathFinderT.exe -s C:\WorkDir\Dataset.pbf -feature C:\WorkDir\Dataset.ms1ft -d C:\WorkDir\Proteins.fasta -o C:\WorkDir /ParamFile:C:\WorkDir\MSPF_MetOx_CysDehydro_NTermAcet_SingleInternalCleavage_ReportTop2.txt
MSPathFinder Mods File
See the Example_Files folder for sample modification definition files.
- .NET 4.7.2
- 2.4 GHz, quad-core CPU
- 16 GB RAM
- Windows 7 or newer
- 250 GB hard drive
Written by Sangtae Kim, Junkap Park, and Chris Wilkins for the Department of Energy (PNNL, Richland, WA) Copyright 2015, Battelle Memorial Institute. All Rights Reserved.
Licensed under the Apache License, Version 2.0; you may not use this file except in compliance with the License. You may obtain a copy of the License at http://www.apache.org/licenses/LICENSE-2.0
RawFileReader reading tool. Copyright © 2016 by Thermo Fisher Scientific, Inc. All rights reserved.
Notice: This computer software was prepared by Battelle Memorial Institute, hereinafter the Contractor, under Contract No. DE-AC05-76RL0 1830 with the Department of Energy (DOE). All rights in the computer software are reserved by DOE on behalf of the United States Government and the Contractor as provided in the Contract. NEITHER THE GOVERNMENT NOR THE CONTRACTOR MAKES ANY WARRANTY, EXPRESS OR IMPLIED, OR ASSUMES ANY LIABILITY FOR THE USE OF THIS SOFTWARE. This notice including this sentence must appear on any copies of this computer software.