Quick Start Tutorial

Basic Workflow

Pioneer performs three major steps:

1. Convert vendor MS files into the Arrow format using PioneerConverter.
2. Build in silico spectral libraries using FASTA files and the Koina server.
3. Search DIA experiments using a spectral library and the MS data files.

Pioneer Converter

Pioneer operates on MS/MS data stored in the Apache Arrow IPC format. Use the bundled PioneerConverter via the CLI to convert Thermo RAW files:

pioneer convert-raw /path/to/raw/or/folder

This subcommand accepts either a single .raw file or a directory of files. See the PioneerConverter repository for additional options such as thread count and output paths.

MzML to Arrow IPC (Sciex)

For mzML-formatted data, use:

pioneer convert-mzml /path/to/mzml/or/folder

Starting Pioneer

After installation, Pioneer is accessed from the command line. Running pioneer --help displays available subcommands:

pioneer [options] <subcommand> [subcommand-args...]

Subcommands include search, predict, params-search, params-predict, convert-raw, and convert-mzml. On the first launch macOS performs a one-time Gatekeeper check.

A minimal end-to-end workflow is:

# Generate library build parameters (library name derived from output path)
pioneer params-predict lib_dir fasta_dir --params-path=predict_params.json

# Edit predict_params.json to customize:
# - Digestion parameters (missed cleavages, modifications, etc.)
# - For multiple FASTA sources, edit fasta_paths array to include directories and/or files
# - Set calibration file if available for automatic m/z range detection

pioneer predict predict_params.json
pioneer convert-raw raw_dir
pioneer params-search library.poin ms_data_dir results_dir --params-path=search_params.json
pioneer search search_params.json

This sequence builds a predicted spectral library, converts vendor files to Arrow, generates search parameters, and searches the experiment.

params = GetBuildLibParams(out_dir, lib_name,
    ["/path/to/uniprot/", "/custom/proteins.fasta"])

params-predict and params-search create template JSON files. Edit these configurations to suit your experiment before running predict or search. See Parameter Configuration for a description of each option.