Abstract
Microbial secondary metabolites represent a rich source for drug discovery, plant protective agents, and biotechnologically relevant compounds. Among them are siderophores, iron-chelating molecules, that show a great influence on bacterial community assembly and the potential to control pathogen invasions. One of such a siderophore is pyoverdine that is produced by fluorescent Pseudomonas members and consists of different peptide chains specific to each bacterial species. The identification and structural elucidation of such suites of siderophores remain widely underexplored as general high-throughput analytical protocols are missing. Therefore, a dedicated method was established allowing a rapid localization and structural elucidation of pyoverdines. Liquid bacterial culture samples were purified by an easy small-scale solid-phase extraction (SPE). Ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) separated highly polar pyoverdines and their derivatives. All ion fragmentation (AIF) generated mass spectra containing the characteristic fragments of the biological precursor of pyoverdine, ferribactin. This led to the revelation of the mass of secreted pyoverdines. Targeted MS/MS experiments at multiple collision energies accomplished the full structure elucidation of the pyoverdine peptide chain. A mass calculator and a fragmentation predictor facilitated greatly the interpretation of MS/MS spectra by providing accurate masses for a straightforward comparison of measured and theoretical values. The method was successfully validated using four well-known pyoverdines with various peptide chains. Finally, the applicability was proven by the analysis of 13 unknown pyoverdines secreted by sampled bacterial cultures. Among these, 4 novel pyoverdine peptide chains were discovered and are herein reported for the first time.Graphical abstract
Highlights
Microorganisms produce a vast number of secondary metabolites that play key roles in microbial physiological processes, and for growth and survival
To avoid clogging injectors and column frits through proteins and to minimize matrix effects [28], a sample preparation protocol based on solid-phase extraction (SPE) was developed
Siderophores gained a large interest in research due to their pathogen control properties making systematic structure elucidation methods at high-throughput desirable
Summary
Microorganisms produce a vast number of secondary metabolites that play key roles in microbial physiological processes, and for growth and survival. Secondary metabolites are low-molecular-weight compounds with various bioactive properties specific to the species that exudes them. The interest for microbial secondary metabolites increased immensely. They serve as a primary source for drug discovery resulting in novel antibiotics, antitumor drugs, or immunosuppressants [6, 7]. Iron-chelating metabolites are utilized for medicinal and agricultural applications [9]. These iron carriers are called siderophores, possess a high iron-binding affinity (> 1 030 M−1), and are with over 500 structures chemically diverse [10].
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