GNPS2 Classical Molecular Networking

Classical molecular networking organizes MS/MS spectra into a graph in which nodes represent consensus fragmentation spectra and edges connect spectra above defined similarity thresholds. The workflow is useful when chromatographic feature tables are unavailable or when the analysis is centered directly on MS/MS spectral relationships.

1. Prepare centroided MS/MS data

   Convert or export tandem mass-spectrometry data in a GNPS-compatible open format and organize files and optional sample metadata for analysis.

2. Select or upload data in GNPS2

   Choose uploaded files or reference public files using Universal Spectrum Identifiers (USIs) in the Classical Molecular Networking workflow.

3. Filter and organize MS/MS spectra

   Apply precursor and fragment tolerances, minimum peak requirements, and networking filters appropriate to the instrument and experiment.

4. Construct the molecular network

   Connect spectra that pass the selected similarity and matched-fragment criteria, producing molecular families of spectrally related ions.

5. Search spectral libraries and inspect networks

   Overlay community spectral-library matches, review singletons and molecular families, and export or visualize network results.

Requirements

• Centroided MS/MS data with precursor information and instrument-appropriate mass tolerances.

Considerations

• Unlike FBMN, classical molecular networking is spectrum-centric and does not inherently preserve chromatographic feature quantification or resolve coeluting/isomeric features through an external feature table.
• Network topology depends on filtering and similarity thresholds; these parameters should be reported together with the GNPS task identifier.

Aron AT, Gentry EC, McPhail KL, et al. Reproducible molecular networking of untargeted mass spectrometry data using GNPS. Nature Protocols 15, 1954–1991 (2020).