Caulobacter lipid A LC-MSMS

Published: 19 May 2022| Version 1 | DOI: 10.17632/y24rjwkb48.1
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Description

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.

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High performance liquid chromatography-tandem mass spectrometry (HPLC-MSMS) of lipid A extracts. All samples were generated by a modified Caroff extraction protocol. Each extract was initially dissolved in 100 µL 1:2 chloroform: methanol before dilution 1:10 with methanol for analysis. A 2-5 µL aliquot of each solution was injected onto a Phenomenex Jupiter C4 column (2 x 50 mm, 5 µm, 300 Å) for HPLC-MSMS analysis with a Waters Acquity UPLC system coupled to a Thermo LTQ-Orbitrap Velos Pro mass spectrometer, equipped with an atmospheric pressure electrospray ionization source. For lipid detection, the HPLC-MSMS analyses were carried out with full-mass detection over a mass range of m/z 250 to 2000 in the Fourier transform MS mode, with negative-ion detection. The mass resolution was 60,000 FWHM @ m/z 400. Fragmentation product ion masses of the three most intense precursor ions were measured in the ion trap or orbitrap (7500 resolution) mass analyzer using stepped collision-induced dissociation (35% of the normalized collision energy) or Higher energy collision-induced dissociation (35% of the normalized collision energy) activation energies. During data acquisitions, real-time mass calibration was applied with m/z 283.26454 as the lock mass for negative-ion detection. The mobile phase for separation was (A) 1 mM ammonium acetate solution and (B) 90% (1:1 acetonitrile/propanol)/10% water/1 mM ammonium acetate as the binary solvents for the 16-minute gradient elution: 0 to 10 min, 30% to 100%B; 10 to 12 min, 100% B and 12 to 12.1 min at 30% B, followed by column equilibration at 30% B from 12.1 to 16 min. The column flow rate was 0.35 mL/min and the column temperature was maintained at 40°C. Hydrophobic interaction liquid chromatography-mass spectrometry (HILIC-MS). A 10-µL aliquot of each solution was injected into a Waters Atlantis HILIC column (4.6 mm x 150 mm, 5 µm) to run LC-MS on a Water Acquity UPLC system coupled to a Thermo LTQ-Orbitrap Velos Pro mass spectrometer, equipped with an atmospheric pressure electrospray ionization source. For lipid detection, the HILIC-MS runs were carried out with full-mass detection over a mass range of m/z 80 to 2000 in Fourier transform MS mode, with positive-ion and negative-ion detection, respectively, in two rounds of LC injections. The mass resolution was 60,000 FWHM @ m/z 400. During data acquisitions, real-time mass calibration was applied with m/z 391.28426 as the lock mass for positive-ion detection and with m/z 112.98563 as the lock mass for negative-ion detection. The mobile phase of HILIC was (A) 20 mM ammonium acetate solution (pH adjusted to 4.0 with acetic acid) and (B) methanol as the binary solvents for gradient elution: 0-4 min, 99% B; 4 to 12.5 min, 99% to 20% B and 12.5 to 15 min at 20% B, followed by column equilibration at 99% B for 5 min between injections. The column flow rate was 0.4 mL/min and the column temperature was maintained at 40°C.

Institutions

University of California Berkeley, University of Victoria Genome British Columbia Proteomics Centre

Categories

Microbiology, Microbial Lipids, Functional Lipid

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