Caulobacter lipid A LC-MSMS

We generated strains of Caulobacter crescentus NA1000 lacking ctpA or lpxC, performed an extraction of free lipid A, and examined the samples by liquid chromatography-tandem mass spectrometry. Details of strain construction and lipid A extraction can be found in the linked article by Zik et al. We analyzed the following strains: wild-type (KR4000), ΔsspB (KR1499), Δfur ΔsspB (KR4077) ΔctpA Δfur ΔsspB (KR4102), and ΔlpxC Δfur ΔsspB (KR4103). KR4000, KR1499, and KR4077 contained lipid A matching the previously determined wild-type lipid A structure. Notably, KR4102 contained no lipid A with sugars at the terminal (1 and 4′) positions but rather contained phosphates, as in the canonical lipid A structure of Escherichia coli. KR4103 strain contained no ions of the wild-type mass but possessed an ion at 1412 m/z, the structure of which remains unclear. The HPLC-MSMS data of this ion showed no loss of phosphate, as seen in KR4102, nor loss of sugars, as seen for KR4000, KR1499, and KR4077. The fragmentation pattern strongly suggested that something other than lipid A was responsible for the ion at 1412 m/z. Given that cardiolipin is a common microbial membrane lipid, we carried out HILIC-MS (described below) with cardiolipin and lipid A standards. Both standards were retained by HILIC, as expected for hydrophobic molecules, but extracts from KR4103 mutant showed no ions at all, suggesting that the species at 1412 m/z is not hydrophobic enough to be retained. Regrettably, there remains no structure identified for the ion at 1412 m/z. Structure analysis was conducted manually according to our prior effort in this field (Yoon et al., 2016). Results based on these data are published in biorxiv: https://doi.org/10.1101/2022.01.20.477143.

本研究通过构建缺失 ctpA 或 lpxC 基因的 Caulobacter crescentus NA1000 菌株，提取了游离的脂质 A，并利用液相色谱-串联质谱法对其样本进行了分析。菌株构建和脂质 A 提取的详细过程可参见 Zik 等人发表的关联文章。我们分析了以下菌株：野生型（KR4000）、ΔsspB（KR1499）、Δfur ΔsspB（KR4077）、ΔctpA Δfur ΔsspB（KR4102）以及 ΔlpxC Δfur ΔsspB（KR4103）。KR4000、KR1499 和 KR4077 中检测到的脂质 A 与先前确定的野生型脂质 A 结构相匹配。值得注意的是，KR4102 菌株中在末端（1 和 4′）位置未检测到糖类，而是含有磷酸，这与大肠杆菌的典型脂质 A 结构相似。KR4103 菌株中未检测到野生型质量的离子，但存在一个质量为 1412 m/z 的离子，其结构尚不明确。该离子的 HPLC-MSMS 数据显示，与 KR4102 类似，没有磷酸的损失，也没有如 KR4000、KR1499 和 KR4077 所见的糖类损失。碎片模式强烈暗示，质量为 1412 m/z 的离子并非由脂质 A 导致。鉴于心磷脂是微生物细胞膜中常见的脂质，我们使用心磷脂和脂质 A 标准品进行了高效液相色谱-质谱联用（HILIC-MS）分析（如下所述）。如预期，两者在 HILIC 中均被保留，对于疏水性分子而言，这是典型的现象。然而，KR4103 突变株的提取物中未检测到任何离子，这表明在 1412 m/z 处的物种疏水性不足以被 HILIC 保留。遗憾的是，至今仍未确定质量为 1412 m/z 的离子的结构。结构分析是根据我们在此领域的前期研究（Yoon 等，2016年）手动进行的。基于这些数据的结果已发表在 biorxiv 上：https://doi.org/10.1101/2022.01.20.477143。