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Ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics

Setting up FragPipe

Install or update Java

FragPipe and MSFragger both require a 64-bit Java to run. Download 64-bit Java here by selecting the Windows x64 Installer. Launch the installer and follow the prompts. (You may need to restart FragPipe after updating Java.)

Install or update FragPipe

FragPipe can be downloaded here. Follow the instructions on that same Releases page to launch the program. When FragPipe launches, the first tab in the window (‘Config’) will be used to configure the program.

Install Mono (required for Thermo .raw file reading on Linux)

Linux users need to have Mono installed to read Thermo .raw files.

Install, update, or use an already downloaded version of MSFragger

Use an existing MSFragger .jar file: In FragPipe, use the ‘Browse’ button to select the .jar file.

Update MSFragger: In FragPipe, use the ‘Update’ button to upgrade to the latest version of the MSFragger .jar file. Click ‘Choose file’ to select your current .jar file, and select the latest release (we recommend downloading the zip version, as it can read raw LC-MS files).

Download MSFragger for the first time:

  1. In FragPipe, select the ‘Download’ button to navigate to the download page. On the download page, click the ‘BUY’ button (cost: $0) to choose the offering appropriate for you (academic users should select the second option, all others should select the first).
  2. Fill in your name and email, then review and accept the license terms. Provide the remaining information, and confirm. The bottom of the next page will have a link to download the first MSFragger release. Unzip this compressed file.
  3. Use the ‘Update’ button in FragPipe to upgrade this first release to the latest version. Click ‘Choose file’ to select the .jar file you just downloaded, and select the latest release. The zip version is strongly recommended, as it can read raw LC-MS files. Always keep the original directory structure of the MSFragger download (e.g. do not move the .jar file separately).
  4. In FragPipe, click ‘Browse’ to select the updated .jar file.

Install, update, or use an already downloaded version of Philosopher

If you have already downloaded Philosopher, use the ‘Browse’ button in FragPipe to select the latest Philosopher executable file. To upgrade to the most recent release or to download for the first time, use the ‘Download’ button.

Optional: install, update, or use an already installed version of Python

Database splitting (to reduce the size of the in-memory fragment ion index– helpful for workstations with limited memory or for complex searches) and/or spectral library generation will require Python 3 or above.

If you already have Python 3 or above, make sure the following packages are installed: numpy, pandas, matplotlib, cython, and msproteomicstools. The easypqp package is also required to build spectral libraries from timsTOF data. Please note: if Python was installed through Anaconda, you will already have all of these packages except for msproteomicstools and easypqp. In most cases, you can run pip install [package name] to install a missing package. To install easypqp, you will need to 1) install Git if you don’t already have it, then 2) open an Anaconda Prompt command line window and run these two commands:

pip uninstall --yes easypqp

pip install git+

If Python 3 is not already installed: 1) Click ‘Download’ in the Python section of the Config tab in FragPipe or click here to go to the Anaconda site, click ‘Download’, then select the latest Python version (3.7 or higher) and launch the installer.

2) Follow the prompts in the graphical installer. Note the install location that you choose and complete the installation. We do not recommend adding Anaconda to your PATH environment variable, but you can choose to register Anaconda as your default Python.

3) From the start menu, search for “Anaconda Prompt” and launch it.

4) In the Anaconda Prompt window that opens, type pip install msproteomicstools and hit enter to install the msproteomicstools package. Repeat this for the remaining packages (numpy, pandas, matplotlib, cython). To install easypqp for spectral library generation, you will need to 1) install Git if you don’t already have it, then 2) run these two commands:

pip uninstall --yes easypqp

pip install git+

5) In FragPipe, use the ‘Browse’ button to navigate to the installation location and select python.exe.

Next: see the FragPipe usage tutorial.