MetaMorpheus: Free, Open-Source PTM Discovery

Download the current version here. For first-time Windows users, choose “MetaMorpheusInstaller.msi” and install MetaMorpheus. Check out our getting started playlist on YouTube

MetaMorpheus is a bottom-up proteomics database search software with integrated post-translational modification (PTM) discovery capability. This program combines features of Morpheus and G-PTM-D in a single tool.

Check out the wiki page for software details!

Major Features

• Database Search: A robust search algorithm that identifies peptides by their fragmentation spectra. Watch our Search task YouTube video
• Calibration: A calibration tool that uses peptides identified by a database search to calibrate the m/z values of all peaks in the spectra. This improves the quality of any subsequent search or analysis of the data. Watch our calibration task YouTube video.
• G-PTM-D: Post-translational modification (PTM) discovery, which expands the scope of peptide identifications to include both known and unknown PTMs. Watch our GPTMD task YouTube video.
• Quantification: Ultrafast label-free peptide quantification with FlashLFQ. MS2-identified peptides are used as “seeds” for peakfinding, including PTM-containing peptides. Watch our Label-free quantification with MetaMorpheus video on YouTube.
• O-glycopeptide Characterization: O-Pair Search identifies O-glycopeptides using an ion-indexed open modification search and localizes O-glycosites using graph theory and probability-based localization.

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 the installer (MetaMorpheusInstaller.msi) only works on Windows. The command-line version of MetaMorpheus supports any operating system that supports .NET Core (Windows, MacOS, Linux)
• 8 GB RAM recommended

Spectra Requirements

• One of the following formats:
  • Thermo .raw (Windows and Linux only)
  • .mzML file in centroid mode. Please watch our How to convert files to .mzML video on YouTube.
  • .mgf
• MS1 and MS2 scans
• If you would like to know more about the types of files that can be searched with MetaMorpheus, please watch our Mass Spectra Files Video on YouTube.

Database Requirements

UniProt .XML or .fasta format; may be used in compressed (.gz) format. If you would like to know how to obtain a UniProt .XML databases, please watch our Protein Databases Video on YouTube.

Getting Started

Please check out our wiki for useful information and guides.

Installation and typical usage is described for the on the Getting Started page:

• Test Installation (Windows GUI)
• Typical Usage (Windows GUI)
• Test Installation (Windows Command Line Executable)
• Test Installation (via .NET Core .dll - Linux, macOS, Windows)
• Test Conda Installation (Linux, macOS, Windows)

References & Citation Guide for MetaMorpheus

MetaMorpheus:

• MetaMorpheus: Enhanced Global Post-translational Modification Discovery with MetaMorpheus, J Proteome Res 2018, 17, 1844-1851.
• Morpheus: A Proteomics Search Algorithm Specifically Designed for High-Resolution Tandem Mass Spectra, J Proteome Res 2013, 12, 1377–1386

GPTMD searches:

• Global Post-Translational Modification Discovery, J Proteome Res 2017, 16, 1383–1390
• Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search, J Proteome Res, 2015, 14, 4714–4720

Quantification:

• Ultrafast Peptide Label-Free Quantification with FlashLFQ, J Proteome Res 2018, 17, 386–391.
• If you use SILAC quantification: An atlas of protein turnover rates in mouse tissues, Nat Communications 2021, 12, 6778.

Crosslinking MS (XL-MS) search: Identification of MS-Cleavable and Noncleavable Chemically Cross-Linked Peptides with MetaMorpheus , J. Proteome Res. 2018, 17, 7, 2370–2376.

Multiple protease parsimony: Improved Protein Inference from Multiple Protease Bottom-Up Mass Spectrometry Data, J Proteome Res 2019, 18, 9, 3429–3438.

Glycoproteomic searches: O-Pair Search with MetaMorpheus for O-glycopeptide characterization, Nat Methods 2020, 17, 1133–1138.

Proteogenomic database searches with Spritz: Spritz: A Proteogenomic Database Engine, J Proteome Res 2021, 20, 1826–1834.

Long-read proteogenomic characterization: Enhanced protein isoform characterization through long-read proteogenomics, Genome Biology 2022, 23, 69.

Spectral library GPTMD search: A Hybrid Spectral Library and Protein Sequence Database Search Strategy for Bottom-Up and Top-Down Proteomic Data Analysis, J of Proteome Res 2022, 21, 2609-2618

Spectral Averaging: Spectral averaging with outlier rejection algorithms to increase identifications in top-down proteomics

mzLib, an all-purpose mass spectrometry toolchest implemented by MetaMorpheus

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.