Structural Elucidation of a Unique Glycerophospho Lipid A From Phenol‐Phase Soluble Lipopolysaccharide of Vibrio anguillarum Serovar SJ‐41

ABSTRACT Rationale In this study, the chemical structure of a unique lipid A derived from the phenol‐phase soluble lipopolysaccharide (LPS) of Vibrio anguillarum serovar SJ‐41, a virulent marine and freshwater pathogen, was investigated using electrospray ionization with field asymmetric wave ion mobility‐Orbitrap mass spectrometry (ESI‐FAIMS‐MS). The analysis indicated that the obtained lipid A consisted of a heterogeneous mixture of molecules. High‐energy collision dissociation tandem mass spectrometry (HCD‐MS/MS) allowed the identification of unique chemical motifs of V. anguillarum lipid A. Methods ESI‐FAIMS‐MS, HCD‐MS/MS, and Kendrick mass defect (KMD) plots were used to elucidate V. anguillarum lipid A molecular structure and mixture heterogeneity. Results Structural analysis revealed significant deviations from canonical lipid A, including the presence of a phospho‐glycerol moiety located on primary acyl chain at the O‐3 position of reducing sugar end and presence of novel di‐hydroxylated primary acyl chains on the N‐2′ position of the non‐reducing sugar end. As far as we know, this is the first report of lipid A structures containing both phospho‐glycerol moiety and di‐hydroxylated primary acyl chains. KMD plots were employed to investigate structural diversity of lipid A complex mixture. Conclusions Tandem mass spectrometric analyses and KMD plots allowed the determination of lipid A structural diversity. These findings provide new insights into the lipid A composition of V. anguillarum strain SJ‐41 and underscore the need for further studies to explore its biological implications, potentially reshaping our understanding of lipid A and its role in host‐pathogen interactions.