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FragPipe is a comprehensive computational platform for analyzing mass spectrometry-based proteomics data. FragPipe comes with an easy to use Java Graphical User Interface (GUI) but can also be run in the command line mode, on Windows, Linux, or in the cloud environment. It is powered by MSFragger - an ultrafast proteomic search engine suitable for both conventional and “open” (wide precursor mass tolerance) peptide identification. FragPipe includes Percolator and the Philosopher toolkit for downstream post-processing of MSFragger search results (PeptideProphet, iProphet, ProteinProphet), FDR filtering, label-based quantification, and multi-experiment summary report generation. FragPipe includes MSBooster module for deep-learning based rescoring of peptide identifications. Crystal-C and PTM-Shepherd are included to aid interpretation of open search results. Also included in FragPipe binary are TMT-Integrator for TMT/iTRAQ isobaric labeling-based quantification, IonQuant for label-free quantification with FDR-controlled match-between-run (MBR) functionality, spectral library building with EasyPQP, and MSFragger-DIA, DIA-Umpire SE, and diaTracer modules for direct (“library-free”) analysis of data independent acquisition (DIA) data. FragPipe includes DIA-NN for extraction of quantification from DIA data.


Docker image

FragPipe tutorials


Using FragPipe with other tools

Supported instruments and file formats

The table below shows the compatibility of FragPipe workflow components with different spectral file formats.

Bruker .d indicates ddaPASEF files from timsTOF, other Bruker .d files should be converted to .mzML. Please also note that timsTOF 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.

Workflow Step .mzML Thermo (.raw) Bruker (.d) .mgf
DIA-Umpire pseudo-MS/MS generation    
diaTracer pseudo-MS/MS generation      
MSFragger search
Crystal-C artifact removal    
PTMProphet localization  
PTM-Shepherd summarization  
Label-free quantification  
SILAC/dimethyl quantification  
TMT/iTRAQ quantification    
Spectral library generation
DIA-NN quantification ✔*  

DIA data acquired with overlapping/staggered windows must be converted to mzML with demultiplexing. Quantification from Thermo .raw files with DIA-NN requires installation of Thermo MS File Reader, see the DIA-NN documentation for details.

Please note TMT/iTRAQ quantification from Thermo .raw files will take longer than from .mzML files.

Additional Documentation

Complete MSFragger documentation can be found on the MSFragger wiki. For documentation on the Philosopher toolkit see the Philosopher wiki.

Questions and Technical Support

View previous questions/bug reports in the FragPipe issue tracker. Please post any new questions/bug reports regarding FragPipe itself here as well. For questions specific to individual components of FragPipe you can also use MSFragger issue tracker, Philosopher issue tracker, IonQuant issue tracker. See the MSFragger wiki and FAQ.

For other tools developed by Nesvizhskii lab, visit our website

How to Run


FragPipe is open source and the output is currently supported by the following software projects:

Key references

DDA quantification

Building from scratch

  1. Update build version:
    The version of the build is stored in 3 separate places:
    • File: MSFragger-GUI/src/umich/msfragger/gui/
      Property: msfragger.gui.version
    • File: MSFragger-GUI/build.gradle
      Property: version
    • File: MSFragger-GUI/src/umich/msfragger/gui/
      Property: msfragger.gui.version
  2. Build:
    You don’t need to have Gradle installed, the Gradle wrapper included in this repository will be used. From the root directory of the repository issue the following commands:

     cd ./MSFragger-GUI
     ./gradlew makeReleaseZipNoJre

    or use this version to build with Java Runtime (for Windows only):

     cd ./MSFragger-GUI
     ./gradlew makeReleaseZipWithJre
  3. The .zip output will be in MSFragger-GUI/build/github-release.