A Multimodal System for Lipid A Structural Analysis from a Single Colony

Structural elucidation of Gram-negative bacterial lipid A traditionally requires chemical extraction followed by tandem MS data in the negative ion mode. Previously, we reported FLAT and FLATⁿ as methods to rapidly determine the structure of lipid A without chromatographic techniques. In this work, we extend the capability and effectiveness of these techniques to elucidate the chemical structure in a de novo manner by including the use of positive ion mode (FLAT⁺ and FLATⁿ⁺) spectral approaches. Advantages of positive mode analysis of lipid A include the generation of more interpretable and informative fragmentation patterns that include the identification of diagnostic fragments, including selective dissociation of a glycosidic bond between two glucosamine units and the selective dissociation at the secondary acyl chain in 2'-N, allowing for the determination of the composition of fatty acids. As a proof of principle, we present here two previously uncharacterized structures of lipid A from Roseomonas mucosa (R. mucosa) and Moraxella canis (M. canis). In R. mucosa, we determined the lipid A structure with a nonconventional backbone of-β-1,6 linked 2,3-dideoxy-2,3-diamno-d-glucopyranose further modified with galacturonic acid in the place of typical 1-phosphate, and in M. canis, we assigned a single discrete structure using the specific fragmentation patterns of terminal phosphate groups present in lipid A. Therefore, FLATⁿ⁺, in combination with FLAT and FLATⁿ, provides a multimodal structural platform for rapid structure characterization of unusual and complex lipid A structures from a single colony.