Metal chelating small molecules (metallophores) play significant roles in microbial interactions and bacterial survival
however, current methods to identify metallophores are limited by low sensitivity, a lack of metal selectivity, and/or complicated data analysis. To overcome these limitations, we developed a novel approach for detecting metallophores in natural product extracts using ion mobility-coupled mass spectrometry (IM-MS). As a proof of concept, marine bacterial extracts containing known metallophores were analyzed by IM-MS with and without added metals, and the data were compared between conditions to identify metal-binding metabolites. Ions with changes in both mass and mobility were specific to metallophores, enabling their identification within these complex extracts. Additionally, we compared the use of direct infusion (DI) and liquid chromatography (LC) separation with IM-MS. For most samples, DI outperformed LC by minimizing the time required for data collection and simplifying analysis. However, for some samples, LC improved the detection of metallophores likely by reducing ion suppression. IM-MS was then used to identify 10 metallophores in an extract from a marine