Structural characterization of multiple pyoverdines secreted by two Pseudomonas strains using liquid chromatography-high resolution tandem mass spectrometry with varying dissociation energies.

High-affinity iron (Fe)-scavenging molecules, or siderophores, are secreted by microorganisms to acquire and compete for Fe. Pyoverdine (PVD), the primary siderophore produced by Pseudomonas, consists of a dihydroxyquinoline-type chromophore, a peptide chain of variable length and conformation, and a side chain composed of a dicarboxylic acid or its monoamide derivative. Elucidation of the PVD structures secreted by different Pseudomonas strains is an important step toward understanding their Fe-transport strategies. In this study, we characterized multiple PVDs secreted by Pseudomonas putida KT2440 and Pseudomonas fluorescens RA12 using ultra-high performance liquid chromatography coupled with high-resolution quadrupole-orbitrap tandem mass spectrometry. To avoid purification steps prior to characterizing the bacterial supernatants, PVD candidates were identified by extracting fragments of the dihydroxyquinoline component from the chromatographic peaks. Varying collisional dissociation energies were subsequently applied to achieve, with high mass accuracy, a broad coverage of fragments of the entire PVD. Our approach allowed us to discriminate between three different PVD structures in the secretion of each strain. The three PVDs of P. putida possess the same peptide chain of seven amino acids, Asp-Orn-OHAsp-Dab-Gly-Ser-cOHOrn, with a cyclicized portion present in two of the PVDs. For P. fluorescens, two of the PVDs had the same peptide chain of 13 amino acids, Ala-Lys-Gly-Gly-Ala-OHAsp-Gly-Ser-Ala-Ala-Ala-Ala-cOHOrn, whereas a third PVD had a Ser substituting for the first Ala. The side chain of the PVDs was either succinic acid or succinamide. The present approach can be employed for simultaneous structural characterization of several peptidic siderophores and related molecules in bacterial secretions. Graphical abstract Characterizing mutiple pyoverdine (PVD) structures in bacterial secretions without prepurification step.