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.