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MSFragger

Ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics

MSFragger

MSFragger is an ultrafast database search tool for peptide identification in mass spectrometry-based proteomics. It has demonstrated excellent performance across a wide range of datasets and applications. The speed of MSFragger makes it particularly suitable for the analysis of large datasets (including TIMS-TOF PASSEF data), for enzyme unconstrained searches (e.g. peptidome), for ‘open’ database searches (with the precursor mass tolerance set to hundreds of Daltons) for identification of modified peptides, and for glycopeptide identification (N-linked and O-linked) with MSFragger Glyco mode.

MSFragger is implemented in the cross-platform Java programming language, and can be used three different ways:

  1. With FragPipe GUI (Graphical User Interface).
  2. Through ProteomeDiscoverer
  3. As a standalone Java executable (JAR) file

MSFragger writes output in either tabular or pepXML formats, making it fully compatible with downstream data analysis pipelines such as Trans-Proteomic Pipeline and Philosopher. See the complete documentation, including a list of Frequently Asked Questions. Example parameter files can be found here.

Supported instruments and file formats

mzML/mzXML: Data from any instrument in mzML/mzXML format can be used.

Thermo RAW: MSFragger can read Thermo raw files (.raw) directly. FragPipe has limited support for RAW files (Spectral library building is currently not compatible with RAW files), so conversion to mzML is recommended. The MSFragger ProteomeDiscoverer (PD) node is fully compatible with all downstream PD tools.

Bruker TIMS-TOF: MSFragger can read Bruker timsTOF raw files (.d) directly, as well as MGF files converted by Bruker DataAnalysis. Quantification requires .d files.

TIMS-TOF data requires Visual C++ Redistributable for Visual Studio 2017 in Windows. If you see an error saying cannot find Bruker native library, please try to install the Visual C++ redistibutable.

Download MSFragger

Whether you run use FragPipe, PD, or the command line, you will need to download the latest MSFragger JAR file. See instructions for downloading or upgrading MSFragger.

Release Notes

The latest version of MSFragger was released on 2020-06-05. Check here for the full list of MSFragger versions and changes.

Running MSFragger

FragPipe

On Windows, the easiest way to run MSFragger is through the FragPipe GUI. See this basic usage tutorial and how to convert Thermo RAW files.

FragPipe includes post-database search tool Philosopher (for downstream analysis with PeptideProphet and ProteinProphet), label-free and label-based quantification, FDR filtering, and report generation (at the PSM/ion/peptide/protein-levels). Additional tools include DIA-Umpire SE module for DIA data (currently supporting Thermo data in mzXML format only), Crystal-C for removing open search artifacts, IonQuant for label-free quantification (including match-beetween-runs functionality), TMT-Integrator for iTRAQ/TMT analysis, PTM-Shepherd for generating global PTM profiles, and SpectraST or EasyPQP-based spectral library building module.

ProteomeDiscoverer node

MSFragger and Philosopher (PeptideProphet) are also available as processing nodes in Proteome Discoverer (PD, Thermo Scientific). Currently, the MSFragger-PD node can be used in PD versions 2.2, 2.3 and 2.4.

Please visit our PD-Nodes page for more information.



Command line

See Launching MSFragger on the Wiki page.

Philosopher pipeline

Complete command line analyses can be performed with Philosopher, see this tutorial for a step-by-step example.

Documentation

For technical documentation on MSFragger (hardware requirements, search parameters, etc.), see the MSFragger Wiki page. Tutorials for common MSFragger-related workflows are listed below.

Questions and Technical Support

See our Frequently Asked Questions (FAQ) page. Please post all questions/bug reports regarding MSFragger itself on the MSFragger GitHub page, or if more appropriate on FragPipe page or Philosopher page.

Requests for Collaboration

If you would like to propose a new collaboration that can take advantage of MSFragger and related tools, please contact us directly.

How to Cite

Kong AT, Leprevost FV, Avtonomov DM, Mellacheruvu D, Nesvizhskii AI. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nature Methods 14:513–520 (2017). Manuscript.

For other tools developed by the Nesvizhskii lab, see our website www.nesvilab.org

Disclaimer

The pepXML files produced by MSFragger may have additional attributes (e.g., uncalibrated_precursor_neutral_mass and ion_mobility) not in the original schema. According to our tests, both PeptideProphet and Philosopher can process those additional attributes.