Selective purification of catecholate, hydroxamate and α-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Abstract

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydroxamates, α-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography – mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample ‘contaminants’, therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, dereplication of known features and selection of significant features in the TDAC eluates afforded a fast identification of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in combination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation.