New chromatographic and data evaluation approaches for the comprehensive investigation of metal-phytosiderophore-complex dynamics.

BACKGROUND: Phytosiderophores (PS) are root exudates released by graminaceous plants that play a crucial role in micronutrient acquisition. This is particularly relevant for iron, but PS can also complex other transition metals in the rhizosphere, influencing their bioavailability and uptake. However, characterizing intact metal-PS complexes remains analytically challenging, and studies have largely relied on targeted approaches using a limited number of selected PS.

RESULTS: We developed a liquid chromatography electrospray ionization high resolution mass spectrometry (LC-HRMS) workflow using a mixed-mode stationary phase to separate 2'-deoxymugineic acid, mugineic acid and 3″-epi-hydroxymugineic acid along with their Fe(III), Cu(II) and Ni(II) complexes. Complex integrity was preserved under mild gradient conditions, enabling quantification in negative ionization mode across a working range of 1 to 15 μmol L-1. In parallel, we utilized MetalPicker, a novel software tool for non-targeted detection of metal complexes in LC-HRMS data. By leveraging the distinctive isotopic distribution of transition metals, MetalPicker enables the discovery of metal-PS features in a non-targeted fashion. Validation of MetalPicker against a manufacturer software for targeted quantitative analysis yielded comparable results, demonstrating the accuracy and applicability of MetalPicker for non-targeted metal complex analysis. Our approach was applied for competitive complexation experiments in model samples, revealing a strong affinity of the PS towards Cu(II), potentially limiting iron bioavailability for plants.

SIGNIFICANCE: Our study presents the first chromatographic method separating metal-PS complexes alongside free PS, integrated with the novel non-targeted metal-complex detection software MetalPicker. Together, these advances enable the determination of coexisting metal-PS complexes in samples, expanding analytical capabilities for studying rhizosphere metal dynamics and nutrient mobilization.