Tandem mass spectrometry determination of the putative structure of a heterogeneous mixture of Lipid A s isolated from the lipopolysaccharide of the Gram-negative bacteria Aeromonas liquefaciens SJ-19a Mervt Almostafa 1 , Bashaeer Allehyane 1 , Stefana Egli 1 , Christina Bottaro 1 , Travis D. Fridgen 1 and Joseph Banoub 1,2 * 1 Chemistry Department, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada 2 Special Projects, Science Branch, Department of Fisheries and Oceans Canada, St. John’s, Newfoundland, Canada RATIONALE: We report herein the electrospray ionization mass spectrometry (ESI-MS) and low-energy collision- induced dissociation tandem mass spectrometry analysis (CID-MS/MS) of a mixture of lipid A s isolated from the rough lipopolysaccharide (LPS) of the mutant wild strain of the Gram-negative bacteria Aeromonas liquefaciens (SJ-19a, resistant) grown in the presence of phages. The interaction between the phages and the Gram-negative bacteria regulates host specificity and the heterogeneity of the lipid A component of the LPS. METHODS: The heterogeneous mixture of lipid A s was isolated by the aqueous phenol method from the LPS of the rough wild strain of Gram-negative bacteria Aeromonas liquefaciens (SJ-19a). Hydrolysis of the LPS was with 1% acetic acid, and purification was by chromatography using Sephadex G-50 and Sephadex G-15. ESI-MS and low-energy CID-MS/MS analyses were performed with a triple-quadrupole (QqQ) and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. RESULTS: Preliminary analysis of the lipid A s mixture was conducted by ESI-MS in the negative ion mode and the spectrum obtained suggested that the lipid A SJ-19a was composed of a heterogeneous mixture of different lipid A molecules. CID-MS/MS experiments confirmed the identities of the various mono-phosphorylated β-D-GlcpN-(1→6)- α-D-GlcpN disaccharide entities. This lipid A s mixture was asymmetrically substituted with fatty acids such as ((R)- 14:0(3-OH)), (14:0(3-(R)-(O-12:0)) and (14:0(3-(R)-O-(14:0)) located on the O-3, O-3’, N-2 and N-2’ positions, respectively. CONCLUSIONS: Low-energy collision-induced dissociation tandem mass spectrometry in-space (QqQ-MS/MS) and in-time (FTICR-MS/MS) allowed the exact determination of the fatty acid acylation positions on the H 2 PO 3 →4-O′-β-D-GlcpN-(1→6)- α-D-GlcpN disaccharide backbones of this heterogeneous mixture of lipid A s , composed inter alia of seven different substituted lipid A s , formed from the incomplete biosynthesis of their respective LPS. Copyright © 2016 John Wiley & Sons, Ltd. Gram-negative bacteria are well known to cause many serious human and animal diseases. [1–3] Typical Gram-negative bacterial envelopes are made by amphiphillic lipopolysaccharide biomolecules known as LPS (also called endotoxin). The molecular structure of LPS consists of three domains: the lipid A (hydrophobic moiety) which is glycosylated by the core oligosaccharide, which in turn is glycosylated by the O-specific polysaccharide. The latter is also called the O-antigen which is the hydrophilic moiety of the LPS. [1–3] Lipid A is the innermost of the LPS molecule and its hydrophobic nature allows it to anchor to the bacterial outer membrane. Lipid A is required for the toxicity of Gram-negative bacteria. [3–5] The O-antigen is composed of repeating oligosaccharide units extending out from the bacteria, whose structure and composition may be different for various geneses and bacterial serotypes. [6–8] The presence or absence of O-antigen determines whether the LPS is considered rough or smooth. Full-length O-chains would render the LPS smooth, whereas the absence of O-chains would make the LPS rough. Semi-rough LPSs are composed of one O-chain unit attached to the core-lipid A oligosaccharide. [6–8] Lipid A is usually involved in the activation of cell-binding proteins such as Toll-like receptor 4 (TLR4)-MD-2 complexes which are responsible for the immune-stimulatory activity. [9–11] The structure of lipid A can be differentiated by the number, length and distribution of the acyl chains on the O-3, O-3′, N-2 and N-2′ positions of the β-D-GlcpN- (1→6)-D-GlcpN disaccharide backbone; and these features are often used as the basis for distinguishing and classifying Gram-negative bacteria. Lipid A is usually phosphorylated at both the O-1 and the O-4′ position of the β-D-GlcpN- (1→6)-α-D-GlcpN disaccharide backbone. [9–11] Aeromonas liquefaciens is a genus of Gram-negative, anaerobic, non-spore-forming bacilli bacterium belonging to the Gram-negative Vibrionaceae family. [12,13] This bacterium fits in a group of closely related motile Aeromonads, which * Correspondence to: J. H. Banoub, Fisheries and Oceans Canada, Science Branch, Special Projects, St. John’s, NL, A1C 5X1, Canada. E-mail: joe.banoub@dfo-mpo.gc.ca Copyright © 2016 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016, 30, 1043–1058 Research Article Received: 1 December 2015 Revised: 3 February 2016 Accepted: 12 February 2016 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2016, 30, 1043–1058 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7540 1043