Analyzing DIA data

FragPipe can be downloaded here. Follow the instructions on that same Releases page to launch the program. See here for help configuring FragPipe.

FragPipe currently offers two workflows for DIA data:

1) DIA_SpecLib_Quant - takes DIA data (plus optional DDA data) as input, builds a spectral library using MSFragger-DIA, then quantifies with DIA-NN

2) DIA_DIA-Umpire_SpecLib_Quant - takes DIA data (plus optional DDA data) as input, DIA-Umpire generates pseudo-MS/MS spectra from the DIA files (instead of direct search with MSFragger-DIA), then MSFragger in DDA mode is used to search, followed by quantification with DIA-NN

Recommendation: do not assign the files to any experimental groups. FragPipe builds a single spectral library using all files.

If more than one experimental group are created, FragPipe will generate a spectral library for each group, and use it for the DIA runs in the same group. Spectral libraries from different groups will not be used together.

Please note:

Configure FragPipe

Python (with EasyPQP installed) is needed for spectral library generation. On the Config tab, check that a valid Python path is provided (Python version will be shown) and that EasyPQP is ‘Available’. If Python is installed but EasyPQP is missing, click the ‘Install/Upgrade EasyPQP’ button and wait a minute or so for installation. For help installing Python, see this page.


  1. In the Workflow tab, select the ‘DIA_SpecLib_Quant’ workflow from the dropdown menu and click ‘Load’.
  2. Load DIA (and optionally additional DDA) spectral files in mzML or raw format. You can use the ‘Add folder recursively’ button to browse for the unzipped ‘speclib-raw’ folder, which will load 2 DIA files and 2 DDA files. The data type of each file will be automatically guessed by FragPipe. The guess is based on the folder and file name. Please double check the data type and adjust it if necessary.

  1. On the Database tab, use ‘Browse’ to select the FASTA sequence database file 2021-05-13-decoys-UPS-reviewed-contam-UP000000589.fas in the ‘speclib-raw’ folder. This is a mouse database with reviewed sequences, decoys, common contaminants, and iRT peptides. UPS protein sequences have been manually added.

  1. On the ‘Quant (DIA)’ tab, note that DIA-NN will be run unless unchecked. The spectral library generated by FragPipe will automatically be passed to DIA-NN for quantification of the DIA files provided.

  1. On the ‘Run’ tab, set the output directory and click ‘Run’.


Follow the same steps as above to run the ‘DIA_DIA-Umpire_SpecLib_Quant’ workflow, which uses DIA-Umpire to generate pseudo-DDA spectra instead of MSFragger-DIA direct search of the DIA files. On the ‘Umpire’ tab, choose the appropriate settings:

Please note that if DIA-Umpire fails or is interrupted, temporary files will cause issues if the process runs again. Make sure to delete any temporary files that are generated alongside the raw/mzML files before re-running FragPipe.

Key References

Yu F, Teo GC, Kong AT, Li GX, Demichev V, Nesvizhskii AI. One-stop analysis of DIA proteomics data using MSFragger-DIA and FragPipe computational platform, biorxiv (2022).

Tsou CC, Avtonomov D, Larsen B, Tucholska M, Choi H, Gingras AC, Nesvizhskii AI. DIA-Umpire: comprehensive computational framework for data-independent acquisition proteomics, Nature Methods 12:258-64 (2015).

Demichev V, Szyrwiel L, Yu F, Teo GC, Rosenberger G, Niewienda A, Ludwig D, Decker J, Kaspar-Schoenefeld S, Lilley KS, Mülleder M, Nesvizhskii AI, Ralser M. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts, Nature Communications 13:3944 (2022).

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