Identification and structural characterization of lipid A from Escherichia coli, Pseudomonas putida and Pseudomonas taiwanensis using liquid chromatography coupled to high-resolution tandem mass spectrometry

Abstract

Rationale: Lipid A is a part of the lipopolysaccharide layer, which is a main component of the outer membrane from Gram-negative bacteria. It can be sensed by mammalians to identify the presence of Gram-negative bacteria in their tissues and plays a key role in the pathogenesis of bacterial infections. Lipid A is also used as an adjuvant in human vaccines, emphasizing the importance of its structural analysis.

Methods: In order to distinguish and characterize various lipid A species, a liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) method was developed. Isolation of lipid A from different bacteria was carried out using a modified Bligh and Dyer extraction following a mild acid hydrolysis. Chromatography was performed using a bifunctional reversed-phase-based stationary phase. High-resolution MS using negative electrospray ionization was applied and MS/MS experiments utilizing high-energy collisional dissociation generated diagnostic product ions, which allowed the assignment of the side chains to distinct positions of the lipid A backbone.

Results: The method was applied to lipid A isolations of Escherichia coli (E. coli), Pseudomonas putida (P. putida) and Pseudomonas taiwanensis (P. taiwanensis). Various lipid A species were identified by their accurate masses and their structures were characterized using MS/MS experiments. Previously described lipid A structures from E. coli were identified and their structures confirmed by MS/MS. For the biotechnologically relevant strains P. putida and P. taiwanensis, we confirmed species by MS/MS, which have previously only been analyzed using MS. In addition, several lipid A species were discovered that have not been previously described in the literature.

Conclusions: The combination of LC and MS/MS enabled the selective and sensitive identification and structural characterization of various lipid A species from Gram-negative bacteria. These species varied in their substituted side chains, speaking of fatty acids and phosphate groups. Characteristic product ions facilitated the assignment of side chains to distinct positions of the lipid A backbone.