Informed Proteomics

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.

Manuscript

Implementation details are described in the manuscript "Informed-Proteomics: open-source software package for top-down proteomics", published in Nature Methods

• PMID 28783154
• Manuscript text in PMC
• DOI 10.1038/nmeth.4388

Install/Tutorials

See the Informed Proteomics GitHub wiki for usage, tutorials, and more!

Downloads

https://github.com/PNNL-Comp-Mass-Spec/Informed-Proteomics/releases

Continuous Integration

The latest versions of the Informed Proteomics tools may be available on the AppVeyor CI server, though they get auto-deleted after 6 months.

MSPathFinderT

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.

Processing steps:

1. 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.

2. 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.

3. 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.

4. Optionally use ProMexAlign to align MS1 features between datasets

• Creates a .tsv file listing a consolidated list of MS1 features, plus the corresponding Feature IDs from the input files
• A FeatureID of 0 means the feature was not present

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:\WorkDir -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.

Running on Linux

PbfGen, ProMex, and MSPathFinderT can be run on Linux using Mono

Example command lines:

mono PbfGen.exe -s *.raw
mono PbfGen.exe -s *.mzML
mono PbfGen.exe -s *.mzML.gz

mono ProMex.exe -i *.pbf -minCharge 2 -maxCharge 60 -minMass 2000 -maxMass 50000 -score n -csv n -maxThreads 0

mono MSPathFinder/MSPathFinderT.exe -s *.pbf -d ID_006407_8F27399B.fasta -o . -ParamFile MSPF_MetOx_CysDehydro_NTermAcet_SingleInternalCleavage.txt

PbfGen Syntax

PbfGen.exe
    -s:RawFilePath (*.raw or directory)
    [-o:OutputDir]
    [-start:StartScan] [-end:EndScan]

-i or -s or -InputFile

• Input file or input directory
• Supports .raw, .mzML, and several vendor formats (see documentation)

-o or -OutputDirectory

• Output directory.
• Default: directory containing input file

-start

• Start scan number
• Optionally use to limit scan range included in .pbf file

-end

• End scan number
• Optionally use to limit scan range included in .pbf file

ProMex Syntax

ProMex.exe 
  -i:InputFile [-o:OutputDirectory]
  [-MinCharge:Value] [-MaxCharge:Value]
  [-MinMass:Value]   [-MaxMass:Value]
  [-FeatureMap:Flag] [-Score:Flag]
  [MaxThreads:Value] [-csv:Flag]
  [BinResPPM:Value]  [ScoreTh:Value]

-i or -s or -InputFile

• Input file or input directory
  • Supports .pbf, .mzML, and several vendor formats (see documentation)

-o or -OutputDirectory

• Output directory.
• Default: directory containing input file

-MinCharge

• Minimum charge state.
• Default: 1
• Min: 1, Max: 60

-MaxCharge

• Maximum charge state
• Default: 60
• Min: 1, Max: 60

-MinMass

• Minimum mass, in Daltons
• Default: 2000
• Min: 600, Max: 100000

-MaxMass

• Maximum mass, in Daltons
• Default: 50000
• Min: 600, Max: 100000

-FeatureMap

• Output the feature heatmap. Defaults to true.
• To disable, use -FeatureMap:false or include 'FeatureMap=False' in a parameter file

-Score

• Output extended scoring information
• Default: False

-MaxThreads

• Max number of threads to use
• Default: 0, meaning automatically determine the number of threads to use

-csv

• Also write feature data to a CSV file
• Default: False

-BinResPPM or -BinningResolutionPPM

• Binning resolution, in ppm
  • This is used when finding LC-MS features
  • Data points whose mass difference is less than the resolution are grouped together
• Allowed values are 1, 2, 4, 8, 16, 32, 64, or 128
• Default: 16

-ScoreTh or -ScoreThreshold

• Likelihood score threshold
• Default: -10

-ms1ft

• Name of a ms1ft feature file to use to create a feature plot (the corresponding .pbf file must be included in the same directory)
• Use -ms1ft. to infer the name from the pbf file

-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.

Create PNG using existing ms1ft file

Command to create a PNG of the features in an existing ms1ft file (requires both a .pbf file and a .ms1ft file):

ProMex.exe -i dataset.pbf -ms1ft dataset.ms1ft -featureMap

ProMexAlign syntax

ProMexAlign.exe DatasetInfoFile

• The dataset info file is a tab-delimited text file with either 3 or 4 columns of information
  • Expected columns:
  • Label RawFilePath Ms1FtFilePath MsPathfinderIdFilePath
• If data in the Label column is an empty string, dataset labels will be auto-assigned as Dataset_1, Dataset_2, etc.
• The raw files are either Thermo .raw files or .pbf files created by PbfGen
• The MsPathfinderIdFilePath column is optional
  • If _IcTda.tsv files are listed in this column, additional columns will appear in the output file created by ProMexAlign

Example input file:

Label	RawFilePath	Ms1FtFilePath	MsPathfinderIdFilePath
Intact_Run8	Intact_100ng_Run8.pbf	Intact_100ng_Run8.ms1ft
Intact_Run9	Intact_100ng_Run9.pbf	Intact_100ng_Run9.ms1ft
Intact_Run10	Intact_100ng_Run10.pbf	Intact_100ng_Run10.ms1ft

MSPathFinder Syntax

MSPathFinderT.exe
  -i:InputFile [-d:FastaFile] 
  [-o:OutputDirectory] [-overwrite:Flag]
  [-mod:ModificationFilePath]
  [-ic:InternalCleavageMode]
  [-tda:DatabaseSearchMode]
  [-tagSearch:Flag]
  [-t:PrecursorTolerance] 
  [-f:FragmentIonTolerance]
  [-MemMatches:Count] 
  [-n:NumMatchesPerSpec]
  [-IncludeDecoys:Flag] 
  [-MinLength:Value] [-MaxLength:Value]
  [-MinCharge:Value] [-MaxCharge:Value]
  [-MinFragCharge:Value] [-MaxFragCharge:Value]
  [-MinMass:Value] [-MaxMass:Value]
  [-Feature:FeatureFile]
  [-Threads:Value]
  [-act:ActivationMethod]
  [-ScansFile:MS2ScanFilterFile]
  [-flip:Flag]
  [-ParamFile]
  [-CreateParamFile]

-i or -s or -specFile

• Spectrum File (.raw or .pbf)

-d or -database

• Database File (*.fasta or *.fa or *.faa)

-o or -outputDir

• Output Directory

-ic

• Internal Cleavage Search Mode
• Default: SingleInternalCleavage (or 1)
• Possible values:
  • 0 or 'NoInternalCleavage': No Internal Cleavage
  • 1 or 'SingleInternalCleavage': Single Internal Cleavage
  • 2 or 'MultipleInternalCleavages': Multiple Internal Cleavages

-TagSearch

• Enable/disable Tag-based Search
• Use true or false
  • Or use '0' for false or '1' for true
  • Or use 'n' for false or 'y' for true
• Default: True

-MemMatches

• Number of matches to keep in memory
  • These matches are used when computing spectral E-values
• Default: 3

-n or -NumMatchesPerSpec

• Number of results to report for each mass spectrum
• Default: 1

-IncludeDecoy or -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; can be 0, 1, or -1
  • 0: target search only
  • 1: target and shuffled decoy database
  • -1: only search shuffled decoy database
• Default: 0

-Overwrite

• Overwrite existing results
• If false, looks for files _IcTarget.tsv and _IcDecoy.tsv and uses the results
• Default: False

-t or -PrecursorTol or -PMTolerance

• Precursor Tolerance (in PPM)
• Default: 10

-f or -FragmentTol or -FragTolerance

• Fragment Ion Tolerance (in PPM)
• Default: 10

-MinLength

• Minimum Sequence Length
• Default: 21

-MaxLength

• Maximum Sequence Length
• Default: 500

-MinCharge

• Minimum precursor ion charge
• Default: 2

-MaxCharge

• Maximum precursor ion charge
• Default: 50

-MinFragCharge

• Minimum fragment ion charge
• Default: 1

-MaxFragCharge

• Maximum fragment ion charge
• Default: 20

-MinMass

• Minimum sequence mass in Da
• Default: 3000

-MaxMass

• Maximum sequence mass in Da
• Default: 50000

-Feature

• Path to a .ms1ft, _isos.csv, or .msalign feature file (typically the results from ProMex)
• Leave blank/undefined if processing multiple input files

-threads

• Maximum number of threads, or 0 to set automatically
• Default: 0

-act or -ActivationMethod

• Activation Method; possible values:
  • 0 or 'CID'
  • 1 or 'ETD'
  • 2 or 'HCD'
  • 3 or 'ECD'
  • 4 or 'PQD'
  • 5 or 'UVPD'
  • 6 or 'Unknown'
• Default: Unknown (or 6)

-ScansFile or -ScansFilePath

• Optional text file with MS2 scans to process (tab, comma, or space separated)
• Any integer in the file is assumed to be a scan number to process

-Flip

• If specified, FLIP scoring code will be used (experimental, 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.

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 suggested input file format is centroided .mzML files.

For versions released after February 1, 2019:

• If running PbfGen and ProMex as 64-bit programs, reading from Thermo .raw files is supported via the included RawFileReader DLLs (no additional software is required).

For versions released prior to February 1, 2019:

• If Thermo's MSFileReader is installed, PbfGen and ProMex also support reading from Thermo .raw files (Download here, requires registration to download).

On Windows, several other formats are supported if an appropriate version of ProteoWizard is installed (Download here, make sure the version downloaded matches system architecture)

On Linux, supported input file formats are .raw, .mzML, and .mzML.gz

MSPathFinder Parameter Files

See the Example_Files directory on GitHub 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 directory for sample modification definition files.

System Requirements

Minimum required:

• .NET 4.7.2

Minimum recommended:

• 2.4 GHz, quad-core CPU
• 16 GB RAM
• Windows 7 or newer
• 250 GB hard drive

Contacts

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.
E-mail: [email protected] or [email protected]
Website: https://github.com/PNNL-Comp-Mass-Spec/ or https://panomics.pnnl.gov/ or https://www.pnnl.gov/integrative-omics/

License

Licensed under the Apache License, Version 2.0; you may not use this program 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.