Mass spectrometric characterization of siderophores produced by Pseudomonas taiwanensis VLB120 assisted by stable isotope labeling of nitrogen source
The structures of three previously unknown siderophores produced by the fluorescent, biotechnologically relevant Pseudomonas taiwanensis (P. taiwanensis) VLB120 bacteria were elucidated by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated to high-resolution tandem mass spectrometry (HRMS/MS). They could be verified as iron(III)- and aluminum(III) complexes as well as the protonated molecules of the siderophores formed by in-source fragmentation. The siderophores were separated according to their different acyl side chains and additionally according their central ions. One of the siderophores was identified as pyoverdine with a malic acid (hydroxy succinic acid) amide side chain and a peptide moiety consisting of Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. The other analytes were assigned to an azotobactin with the identical peptide chain linked to the characteristic chromophoric unit and a pyoverdine with a variation in the amino acid sequence. Proline is directly linked to the pyoverdine chromophore instead of ornithine. Acidic and enzymatic hydrolyses were carried out to analyze the individual amino acids. Beside OHAsn, each amino acid of the peptide part was identified by HILIC–HRMS and comparison to authentic standards. Additionally, 15N-labeled cellular supernatants were analyzed by means of HRMS/MS. The results of the MS/MS experiments complemented by accurate mass data facilitated elucidation of the structures studied in this work and provided further confirmation of the three recently described pyoverdines of P. taiwanensis VLB120 (Baune et al. in Biometals 30:589–597, 2017. https://doi.org/10.1007/s10534-017-0029-7).
KeywordsPseudomonas taiwanensis Structure characterization Siderophores Pyoverdines Azotobactin Hydrophilic interaction liquid chromatography (HILIC)
The authors thank HILICON AB (Umeå, Sweden) for providing the iHILIC Fusion columns. We also acknowledge Dr. Benedikt Cramer (Institute for Food Chemistry, University of Münster, Germany) for technical assistance during LTQ Orbitrap measurements and access to the instrument. Support by the German Research Foundation (INST 211/802-1) is also acknowledged.
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