The Protein Digestion Simulator is a utility for processing FASTA files including converting to/from tab delimited text. The GUI also supports FASTA file validation.
Download a .zip file with the installer from:
https://github.com/PNNL-Comp-Mass-Spec/Protein-Digestion-Simulator/releases
The release page also includes a .zip file with ProteinDigestionSimulator.exe
The latest version of the application may be available on the AppVeyor CI server,
but builds are deleted after 6 months.
Otherwise, see the GitHub release page
The Protein Digestion Simulator program can be used to read a text file containing protein or peptide sequences (FASTA format or delimited text) then output the data to a tab-delimited file. It can optionally digest the input sequences using trypsin, partial trpysin, or various other enzymes, and can add the predicted normalized elution time (NET) values for the peptides. It can also validate a FASTA file, testing it against a set of rules that identify common formatting errors. If you need to validate FASTA files in batch mode from the console, download the ValidateFastaFile Program from its Releases Page on GitHub.
As an alternative to digesting the peptides, the software can read in a FASTA file and create a new FASTA file with all of the protein sequences reversed or even randomized. This new file can be the equivalent length of the original file, or can include just a subset of the original file.
The third key feature of this software is the ability to calculate the number of uniquely identifiable peptides within the input file (digested or undigested), using only mass, or both mass and NET, with appropriate tolerances. Note that the NET values are based on a model trained using data from Dr. Richard D. Smith's laboratory at Pacific Northwest National Lab (Richland, WA) and are thus specific to the reversed phase (C18) capillary chromatography system being used.
For more information on the Kangas/Petritis retention time prediction algorithm, please see:
K. Petritis, L.J. Kangas, B. Yan, M.E. Monroe, E.F. Strittmatter, W.J. Qian, J.N. Adkins, R.J. Moore, Y. Xu, M.S. Lipton, D.G. Camp, R.D. Smith. "Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information", Analytical Chemistry, 78, (14), 5026-5039 (2006).
PMID: 16841926
The Protein Digestion Simulator supports the following enzymes:
| Enzyme Name | Cleavage Residues | Exception Residues | Comments |
|---|---|---|---|
| Fully Tryptic | KR | P | Cleave after K or R, but not if K or R is followed by P |
| Fully Tryptic (no Proline Rule) | KR | Cleave after K or R, even if followed by P | |
| Half (Partial) Trypsin | KR | P | |
| Trypsin Plus FVLEY | KRFYVEL | ||
| Trypsin plus Lys-C | KR | P for R | Either after R (no P) or after K (P allowed) |
| Trypsin plus Thermolysin | KRLFVIAM | P for R | |
| Acetic Acid Hydrolysis | D | ||
| Arg-C | R | ||
| Asp-N | D | Cleaves N-terminal to D | |
| Chymotrypsin | FWYL | ||
| Chymotrypsin plus Trypsin | FWYLKR | ||
| CyanBr | M | ||
| Glu-C | ED | ||
| Glu-C, just Glu | E | ||
| Lys-C | K | ||
| PepsinA | FLIWY | P | |
| PepsinB | FLIWY | PVAG | |
| PepsinC | FLWYA | P | |
| PepsinD | FLWYAEQ | ||
| Proteinase K | AEFILTVWY | ||
| Thermolysin | LFVIAM | ||
| No cleavage rule | Cleave after any residue | ||
| Peptide Database | Do not cleavage after any residue | Use to extract peptides in a given mass range |
When using a parameter file to define the enzyme to use for digestion (via parameter DigestionEnzyme),
use the name shown in the second column of the following table:
- As an alternative to enzyme name, you can use the Enzyme ID
| Human-readable Enzyme Name | Enzyme Name For Parameter File | Enzyme ID |
|---|---|---|
| Fully Tryptic | ConventionalTrypsin | 1 |
| Fully Tryptic (no Proline Rule) | TrypsinWithoutProlineException | 2 |
| Half (Partial) Trypsin | KROneEnd | 5 |
| Trypsin plus FVLEY | TrypsinPlusFVLEY | 4 |
| Trypsin plus Lys-C | TrypsinPlusLysC | 21 |
| Trypsin plus Thermolysin | TrypsinPlusThermolysin | 23 |
| Acetic Acid Hydrolysis | AceticAcidD | 20 |
| Arg-C | ArgC | 13 |
| Asp-N | AspN | 14 |
| Chymotrypsin | Chymotrypsin | 7 |
| Chymotrypsin plus Trypsin | ChymotrypsinAndTrypsin | 8 |
| CyanBr | CyanBr | 10 |
| Glu-C | GluC | 9 |
| Glu-C, just Glu | GluC_EOnly | 12 |
| Lys-C | LysC | 11 |
| PepsinA | PepsinA | 16 |
| PepsinB | PepsinB | 17 |
| PepsinC | PepsinC | 18 |
| PepsinD | PepsinD | 19 |
| Proteinase K | ProteinaseK | 15 |
| Thermolysin | Thermolysin | 22 |
| No cleavage rule | NoRule | 0 |
| Peptide Database | TerminiiOnly | 6 |
For example, include this line in a Key=Value parameter file to select partial trypsin
DigestionEnzyme=KROneEnd- Option 1: Download a zip file with the installer from the GitHub release page
- ProteinDigestionSimulator_Installer.zip
- Extract and run ProteinDigestionSimulator_Installer.exe
- Option 2: Download Protein-Digestion-Simulator.zip from the GitHub release page
- Extract the files
- Run ProteinDigestionSimulator.exe to start the GUI
The Protein Digestion Simulator is typically used as a GUI application, but it also can be run from the Windows console, or on Linux using Mono.
Syntax:
ProteinDigestionSimulator.exe
/I:SourceFastaOrTextFile [/F] [/D] [/M:False] [/AD:AlternateDelimiter]
[/O:OutputDirectoryPath] [/P:ParameterFilePath] [/Q]
[/S:[MaxLevel]] [/A:AlternateOutputDirectoryPath] [/R]
The input file path can contain the wildcard character * and should point to a FASTA file or tab-delimited text file
- Gzipped FASTA files are also supported, e.g. E_coli_K12_UniProt.fasta.gz
Use /F to indicate that the input file is a FASTA file
- If
/Fis not used, the format will be assumed to be FASTA only if the filename ends with .fasta or .fasta.gz
Use /D to indicate that an in-silico digestion of the proteins should be performed
- Digestion options must be specified in the parameter file
By default, when converting a FASTA file to a tab-delimited file, monoisotopic mass values are computed for the proteins
- Optionally use
/M:Falseto disable this
If the input file is not a FASTA file, it is assumed to be tab-delimited
- Use
/ADto customize the delimiter (not applicable for FASTA files) - For example, for .csv files use
/AD:,
The output directory path is optional
- If omitted, the output files will be created in the same directory as the input file
The parameter file path is optional
- If included, it should point to a valid Name=Value parameter file
- An example parameter file is available in the Documentation folder on GitHub
When digesting protein sequences, the protein names are shown at the console
- To disable this, use
/Qat the command line or defineQuietMode=Truein the parameter file
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.
- When using
/S, you can redirect the output of the results using/A - When using
/S, you can use/Rto re-create the input directory hierarchy in the alternate output directory (if defined)
Use /L or /Log to log messages to a file
- By default, log files will include the current date, e.g.
ProteinDigestionSimulator_log_2021-10-20.txt - You can define the name of the log file using
/L:LogFileName.txt
The processing options can be specified in a parameter file using /ParamFile:Options.conf or /Conf:Options.conf or /P:Options.conf
- Define options using the format
ArgumentName=Value - Lines starting with
#or;will be treated as comments - Additional arguments on the command line can supplement or override the arguments in the parameter file
Use /CreateParamFile to create an example parameter file
- By default, the example parameter file content is shown at the console
- To create a file named Options.conf, use
/CreateParamFile:Options.conf
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
The Protein Digestion Simulator 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 2021 Battelle Memorial Institute
The NET Prediction DLL is licensed under the Reciprocal Public License v1.5;
for details see file PNNL_NETPrediction_License.pdf
You may obtain a copy of the License at https://opensource.org/licenses/rpl1.5.txt