Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstraße 48, D-48149, Münster, Germany.
Institute of Food Chemistry (170b) , University of Hohenheim, Garbenstraße 28, D-70593, Stuttgart, Germany.
Anal Bioanal Chem. 2024 Oct;416(25):5513-5525. doi: 10.1007/s00216-024-05451-1. Epub 2024 Jul 25.
Plant-pathogenic bacteria are one of the major constraints on agricultural yield. In order to selectively treat these bacteria, it is essential to understand the molecular structure of their cell membrane. Previous studies have focused on analyzing hydrolyzed fatty acids (FA) due to the complexity of bacterial membrane lipids. These studies have highlighted the occurrence of branched-chain fatty acids (BCFA) alongside normal-chain fatty acids (NCFA) in many bacteria. As several FA are bound in the intact phospholipids of the bacterial membrane, the presence of isomeric FA complicates lipid analysis. Furthermore, commercially available reference standards do not fully cover potential lipid isomers. To address this issue, we have developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method with tandem mass spectrometry (MS/MS) to analyze the phospholipids of various plant-pathogenic bacteria with a focus on BCFA containing phospholipids. The study revealed the separation of three isomeric phosphatidylethanolamines (PE) depending on the number of bound BCFA to NCFA. The validation of the retention order was based on available reference standards in combination with the analysis of hydrolyzed fatty acids through gas chromatography with mass spectrometry (GC/MS) after fractionation. Additionally, the transferability of the retention order to other major lipid classes, such as phosphatidylglycerols (PG) and cardiolipins (CL), was thoroughly examined. Using the information regarding the retention behavior, the phospholipid profile of six plant-pathogenic bacteria was structurally elucidated. Furthermore, the developed LC-MS/MS method was used to classify the plant-pathogenic bacteria based on the number of bound BCFA in the phospholipidome.
植物病原细菌是农业产量的主要制约因素之一。为了有针对性地治疗这些细菌，了解其细胞膜的分子结构至关重要。由于细菌膜脂质的复杂性，以前的研究主要集中在分析水解脂肪酸（FA）上。这些研究强调了许多细菌中支链脂肪酸（BCFA）与正常链脂肪酸（NCFA）的存在。由于几种 FA 结合在细菌膜的完整磷脂中，异构 FA 的存在使脂质分析变得复杂。此外，市售的参考标准并不能完全涵盖潜在的脂质异构体。为了解决这个问题，我们开发了一种反相高效液相色谱（RP-HPLC）与串联质谱（MS/MS）联用的方法，用于分析各种植物病原细菌的磷脂，重点是含有 BCFA 的磷脂。研究揭示了根据结合的 BCFA 与 NCFA 的数量，三种异构的磷脂酰乙醇胺（PE）的分离。保留顺序的验证基于可用的参考标准，结合通过气相色谱-质谱（GC/MS）对水解脂肪酸进行分析后的分级。此外，还彻底检查了保留顺序向其他主要脂质类别的可转移性，如磷脂酰甘油（PG）和心磷脂（CL）。利用有关保留行为的信息，对六种植物病原细菌的磷脂谱进行了结构阐明。此外，还使用开发的 LC-MS/MS 方法根据磷脂组中结合的 BCFA 的数量对植物病原细菌进行分类。
