Integrative open workflow for confident annotation and molecular networking of metabolomics MSE/DIA data

Liquid Chromatography coupled with High-Resolution Mass Spectrometry Data-Independent Acquisition (LC-HRMS/DIA) techniques, including MS^E type enable comprehensive analysis, though it poses challenges for data processing with automatic annotation and molecular networking (MN) implementation. This motivated our proposal, herein, we introduce DIA-IntOpenStream, a new integrated workflow combining open-source software to streamline MS^E data handling. It provides in-house custom database construction, allows the conversion of raw MS^E data to a universal format (.mzML), and leverages open software (MZmine 3 and MS-DIAL) advantages for confident annotation and MN data interpretation. This pipeline enhances the accessibility, reliability, and reproducibility of complex MS^E/DIA studies. Previously, reaching MN of MS^E data required vendor software, or other non-universal MS data formats, limiting integrative MS^E data analysis. We pinpoint the novelty, the main strategies and the drawbacks of this topic. As an applicability case, two independent datasets were evaluated: Dataset 1 consisted of plant extracts (60 Ocotea spp. plant extracts). Dataset 2 a publicly available actinobacteria extract spiked with a pool of authentic standards. This user-friendly pipeline enables the adoption of cutting-edge MS tools and provides value to the scientific community. Overall, it holds promise for speeding up metabolite discoveries towards a more collaborative and open environment for research.