Structural characterization of multiple pyoverdines secreted by two Pseudomonas strains using liquid chromatography-high resolution tandem mass spectrometry with varying dissociation energies
Structural characterization of multiple pyoverdines secreted by two Pseudomonas strains using liquid chromatography-high resolution tandem mass spectrometry with varying dissociation energies
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DOI：
10.1007/s00216-015-8659-5
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发表时间：
2015-06-01
影响因子：
4.3
通讯作者：
Aristilde, Ludmilla
中科院分区：
文献类型：
--
作者：
Wei, Hua;Aristilde, Ludmilla
高亲和力铁（Fe）清除分子，或铁载体，由微生物分泌，以获得和竞争铁。绿脓菌荧光素（PVD）是由假单胞菌产生的主要铁载体，由二羟基喹啉型发色团、可变长度和构象的肽链以及由二羧酸或其单酰胺衍生物组成的侧链组成。阐明PVD结构分泌不同的假单胞菌菌株是了解他们的铁运输策略的重要一步。在这项研究中，我们的特点是多个PVDs分泌的恶臭假单胞菌KT 2440和荧光假单胞菌RA 12使用超高效液相色谱结合高分辨率四极杆-轨道阱串联质谱。为了避免在表征细菌上清液之前的纯化步骤，通过从色谱峰提取二羟基喹啉组分的片段来鉴定PVD候选物。随后应用不同的碰撞解离能，以实现高质量精度，广泛覆盖整个PVD的碎片。我们的方法使我们能够区分每种菌株分泌物中的三种不同PVD结构。恶臭假单胞菌的三个PVD具有相同的七个氨基酸的肽链Asp-Orn-OHSp-Dab-Gly-Ser-cOHOrn，其中两个PVD中存在环化部分。对于荧光假单胞菌，两个PVD具有相同的13个氨基酸的肽链，Ala-Lys-Gly-Gly-Ala-OHAsp-Gly-Ser-Ala-Ala-Ala-Ala-cOHOrn，而第三个PVD具有取代第一个Ala的Ser。PVDs的侧链是琥珀酸或琥珀酰胺。本方法可用于细菌分泌物中几种肽铁载体和相关分子的同时结构表征。
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.