ProteaseGuru: Free and Open-Source Tool for In Silico Database Digestion

Download the current version here.

ProteaseGuru is a in silico digestion tool for the planning of bottom-up proteomic experiments. ProteaseGuru allows for the digestion of one or more protein databases with as many proteases as desired. Results of the various proteolytic digests can be visualized with histograms as well as protein sequence coverage maps.

Check out the wiki page for software details!

Getting Started

ProteaseGuru is a Windows desktop application. To run your first in silico digestion:

1. Get the app

• Install the free .NET 10 Desktop Runtime (Windows x64) if you don't already have it.
• Download ProteaseGuru.zip from the latest release and unzip it to a folder you can write to (not Program Files).
• Double-click ProteaseGuru.exe to launch. (Prefer to build it yourself? See Build from source.)

2. Run a digestion in 5 steps

1. Add a database — on the Databases window, click Add (or drag & drop) a UniProt .xml or .fasta file (.gz is fine). Add as many as you like.
2. Set digestion conditions — pick one or more proteases and set your parameters (missed cleavages, min/max peptide length, optional mass range and modifications).
3. Review & run — the Run window summarizes your databases, proteases, parameters, and output location. Click Run.
4. Explore results — when digestion finishes, the Results Summary opens. Use Histograms to compare proteases across the whole proteome, or Protein Search to view per-protein sequence-coverage maps.
5. Export — result tables are written to the output folder automatically; histograms export as PDF + reproducible .csv, and coverage maps export from the Protein Search window.

For a walkthrough of each window, see the wiki Getting Started page.

Command-line runs

The ProteaseGuru.Cli executable runs the digestion pipeline without opening the desktop interface. It accepts one or more .fasta, .fa, or .xml databases (optionally .gz compressed), and writes the same digestion outputs as a GUI run to the specified output directory.

Use a saved DigestionParameters.toml when you need the full GUI configuration, including protease-specific values and modifications. Any explicitly supplied digestion option overrides that value from the TOML for all selected proteases/RNases, while unspecified values and modifications stay as configured in the file:

ProteaseGuru.Cli.exe --database proteins.fasta --output results --params DigestionParameters.toml
ProteaseGuru.Cli.exe --database proteins.fasta --output results-mc3 `
  --params DigestionParameters.toml --missed-cleavages 3

Or supply shared digestion values for all selected proteases/RNases. Values shown below are defaults; --rna enables RNA mode and otherwise the run is protein mode:

ProteaseGuru.Cli.exe --database proteins.fasta --database contaminants.fasta `
  --output results --protease "trypsin|P" --protease "Lys-C|P" `
  --missed-cleavages 2 --min-length 7 --max-length 50 `
  --min-mass -1 --max-mass -1 --detectability-threshold 0.5

For RNA mode, use RNase names and add --rna. Multiple database and protease options can be space-delimited or repeated. In TOML mode the TOML file supplies protease selection; --protease cannot be combined with it. Shared CLI overrides include missed cleavages, min/max length, min/max mass, detectability threshold, and modified-sequence uniqueness (including explicit true or false). Each supplied override applies uniformly to all proteases/RNases in the TOML. Run ProteaseGuru.Cli.exe --help for all options.

Build from source

Requires the .NET 10 SDK and Windows.

git clone https://github.com/smith-chem-wisc/ProteaseGuru.git
cd ProteaseGuru
dotnet build ProteaseGuru.sln -c Release
dotnet run --project ProteaseGuruGui/ProteaseGuru.Gui.csproj

Build and run the CLI with:

dotnet run --project ProteaseGuru.Cli/ProteaseGuru.Cli.csproj -- --help

Or open ProteaseGuru.sln in Visual Studio 2022, set ProteaseGuru.Gui as the startup project, and press F5.

Major Features

• Ability to digest more than one database for application with multi-species samples such as xenografts, virally infected host cells and microbiome samples.
• In silico digestion with as many proteases as desired!
• Ability to define custom proteases for digestion.
• Uniqueness of peptide sequences are determined both within a database and across all databases being analyzed.
• Visualization of whole proteome digestion results is enabled by the generation and ability to export histograms.
• Search for your proteins of interest and view their in silico digestion with a sequence coverage map with PTM and variant annotation!

System Requirements

• Environment:
  • 64-bit operating system
  • .NET Core 8.0:
    • Windows: https://dotnet.microsoft.com/en-us/download/dotnet/thank-you/sdk-8.0.401-windows-x64-installer
    • macOS, x64 Intel processor: https://dotnet.microsoft.com/en-us/download/dotnet/thank-you/sdk-8.0.401-macos-x64-installer
    • macOS, ARM Apple Silicon processor: https://dotnet.microsoft.com/en-us/download/dotnet/thank-you/sdk-8.0.401-macos-arm64-installer
    • Linux: https://learn.microsoft.com/dotnet/core/install/linux?WT.mc_id=dotnet-35129-website
• Note that ProteaseGuru only works on Windows at this time.
• 8 GB RAM recommended

Database Requirements

UniProt .XML or .fasta format; may be used in compressed (.gz) format.

mzLib

mzLib is a nuget package that we created as an all-purpose toolchest for mass-spec data analysis and many of its functions provide the tools for MetaMorpheus. mzLib is freely available for use in mass-spec applications. You do not need to download mzLib separately to run MetaMorpheus; it is already included.

References