Top-down strategies for the structural elucidation of intact gram-negative bacterial endotoxins

Abstract

Re-modelling of lipopolysaccharides, which are the primary constituent of the outer cell membrane of Gram-negative bacteria, modulates pathogenesis and resistance to microbials. Reported herein is the characterization of intact Gram-negative bacterial lipooligosaccharides (LOS) via a new strategy utilizing online liquid chromatography (LC) coupled with ultraviolet photodissociation (UVPD) mass spectrometry. Compared to collision-based MS/MS methods, UVPD and UVPD/HCD promoted a greater array of cleavages within both the glycan and lipid moieties, including C-C, C-N, C-O cleavages in the acyl chains as well as glycosidic and cross-ring cleavages, thus providing the most far-reaching structural characterization of LOS. This LC-MS/MS strategy affords a robust analytical method to structurally characterize complex mixtures of bacterial endotoxins that maintains the integrity of the core oligosaccharide and lipid A domains of LOS, providing direct feedback about the cell envelope architectures and LOS modification strategies involved in resistance host innate immune defense.

Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

“…An alternative to collision-based methods for ion activation/fragmentation is ultraviolet photodissociation (UVPD) [24–26]. As shown previously, UVPD results in structure-specific glycosidic and cross-ring fragments of lipid A and affords richer fragmentation patterns when compared to conventional collisional activation methods [27–35]. Multi-stage MS n strategies have also been important for structural characterization of lipid A [36–38].…”

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility

“…Very few groups have attempted structural analysis of intact LOS, with or without any preliminary chromatographic separation [24,[28][29][30]. Recently, an improved method was introduced for the analysis of intact LOS by negative-ion, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that utilizes a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose [31].…”

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship

“…[136] Advantages to these methods include reduced sample preparation time, increased species and strain specificity, greater clarity with regard to which chemical structures (rather than components of structures) actually exist in the membrane, and a greater overall understanding of how a bacterium survives in a given environment. Regarding the latter, one example is that many marine bacteria have greater net negative charge associated with their membranes.…”

“…Therefore, tandem mass spectra acquired are never produced by a single precursor ion. Recently, more thorough attempts have been made at chromatographic separation of both lipid A[137] and rLPS[136] with great success. Every extract analyzed thus far has been quite heterogeneous.…”