Novel XAD-LC/MS-FBMN-IMS Strategy for Screening <i>Holo-</i>Hydroxamate Siderophores: Siderome Analysis of the Pathogenic Bacterium <i>Tenacibaculum maritimum</i>

Hydroxamate siderophores are key virulence factors in multiple pathogens, and their structures present interesting scaffolds for potential biotechnological applications such as the design of novel antimicrobials. However, their variable and scarce production, their low stability, and occurrence as complex mixtures in natural samples complicate their analysis. Herein, we present a new strategy, named XAD-LC/MS-FBMN-IMS, which enables the analysis of the <i>holo</i>-hydroxamate siderophore composition from microbial cultures by integrating traditional XAD resin extraction, modern Feature-Based Molecular Networking (FBMN) tools, and a novel direct infusion ion mobility mass spectrometry validation approach. Using ferrioxamine as a model, XAD resins demonstrated high efficiency in extracting hydroxamate siderophore metal complexes when compared to other sorbents. This strategy was applied for detecting the siderophores produced by <i>Tenacibaculum maritimum</i>, a relevant pathogenic bacterium in fish aquaculture. This analysis unveiled three families of hydroxamate siderophores, including three known desferrioxamine derivatives along with 17 new putative acyl-desferrioxamine-like structures. Overall, the XAD-LC/MS-FBMN-IMS strategy significantly enhances siderophore and metallophore investigation in microbial cultures, improving our understanding of their production and roles in bacteria and fungi, ultimately facilitating their biotechnological applications.