Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present Feature-Based Molecular Networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. The FBMN method brings quantitative analyses, isomeric resolution, including from ion-mobility spectrometry, into molecular networks.

1Molecular networking has become a key method used to visualize and annotate the chemical space in 2 non-targeted mass spectrometry-based experiments. However, distinguishing isomeric compounds and

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Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.

General conceptualization 48 RS, DP, LFN, MW, PCD conceptualized the idea of IIMN and its integration into GNPS and 49 feature-finding software tools 50 RS, DP, LFN, PCD wrote the manuscript 51 RS, BA, FH, HUH conceptualized the MZmine feature grouping workflow 52 UK, HH provided discussion and feedback on IIMN and the MZmine workflow 53 Development 54 RS developed the IIMN modules in MZmine and the MS 2 spectral library generation modules 55 MW, RS developed the "supplementary edges" format in the FBMN workflow to enable IIMN 56 MW programmed the IIMN workflow on GNPS 57 RS, MW developed the direct submission of MZmine data to run IIMN on GNPS 58 JR, MGA developed the XCMS/CAMERA IIMN integration in R 59 HT developed the MS-DIAL FBMN and IIMN integration 60 KD developed the MS 2 spectral merge function into the export modules for FBMN, IIMN, and 61 SIRIUS, which was coordinated by SB 62 TP, AK provided feedback and help for the development and integration of IIMN in MZmine 63