Article ID Journal Published Year Pages File Type
1201382 Journal of Chromatography A 2013 11 Pages PDF
Abstract

•Novel UHPLC-ToF-MS method for analysis of metallophores (metal-complexing ligands).•No sample preparation is required, and method validation shows satisfactory figures.•Quantification of cis-dioxido-Mo complexes and Fe-complexes with a single UHPLC run.•Application to the quantification of metallophores in aqueous bacterial samples.•Depletion rate of Mo-protochelin was compared with the bacterial uptake of Mo.

Metallophores are a unique class of organic ligands released, for example, by nitrogen fixing bacteria in their environment in order to recruit the micronutrients molybdenum (Mo) and iron (Fe). Mo and Fe are essential cofactors of nitrogenase that reduces atmospheric nitrogen into bioavailable ammonium. Upon release, these bacterial metallophores bind to both metal cations and oxo-anions in the extracellular medium increasing the bioavailability of the metals to the nitrogen fixers, which can subsequently recruit the complexes. The efficient quantification of those metal complexes is crucial for the understanding of the homeostasis of the metal cofactors of nitrogenase (e.g., Fe and Mo), the dynamics of nitrogen fixation and the nitrogen cycle. A novel direct ultra-high-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UHPLC-ToF-MS) was developed to quantify and monitor the production of Fe and Mo complexes of the catecholate metallophores protochelin (Prot) and azotochelin (Azo) in the growth medium of the nitrogen fixer and model organism Azotobacter vinelandii. Chromatographic separations were carried on a reversed C18-phase with a mobile phase ramped from water to acetonitrile spiked with 1 mmol/L ammonium acetate (pH 6.6) to achieve stability of the metal complexes. Linearity for Mo-protochelin and Fe-protochelin was found at the concentration range between 5.0 × 10−8 and 9.0 × 10−7 mol/L with a limit of detection of 2.0 × 10−8 and 3.0 × 10−8 mol/L, respectively. The coefficient of variation of the procedure is in the range from 1.5 to 3.4%. The validation has hence demonstrated that the UHPLC-ToF-MS methodology is a fast, precise, specific, robust, and sensitive approach allowing the direct measurement of metallophores in growth medium without any sample preparation. The UHPLC-ToF-MS methodology was applied to the analysis of metallophores in our laboratory. Under lower Mo concentration, the Mo-protochelin concentration peaks in the middle lag phase, while the Fe-protochelin concentration rises to two maxima at the beginning of the exponential phase and during the stationary phase. The results indicate that the production of metallophores is highly dynamic throughout the growth and has to be monitored with high sensitivity and temporal resolution.

Related Topics
Physical Sciences and Engineering Chemistry Analytical Chemistry
Authors
, , ,