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

Abstract

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 H2 PO3 →4-O'-β-D-GlcpN-(1→6)-α-D-GlcpN disaccharide backbones of this heterogeneous mixture of lipid As , composed inter alia of seven different substituted lipid As , formed from the incomplete biosynthesis of their respective LPS.

Structural investigation by tandem mass spectrometry analysis of a heterogeneous mixture of Lipid An isolated from the lipopolysaccharide of Aeromonas hydrophila SJ‐55Ra

“…It also contains the branched fatty acid (Cl2:0(3‐ O ‐C14:0)) located at N‐2′ (Scheme ). Please note that the anions at m/z 1796.30, 1688.26, 1506.12, and 1359.75 were similar to those described for the mixture of lipid A n isolated from the original strain of Aeromonas liquefaciens SJ‐55R …”

“…In addition, we have previously established that SORI‐CID‐MS/MS (tandem‐in‐time) analysis of a similar heterogeneous mixture of lipid A n resulted in a simple series of product anions which were produced by elimination of the primary fatty acid (3′ε, 3′α and 3α) at the O‐3′ and O‐3 positions and not from the secondary branched fatty acids . Similar results were noticed when conducting high‐energy CID of the same lipid A series with a MALDI‐TOF/TOF‐MS instrument.…”

“…However, the O‐3′ positions of these two different lipid A n mixtures are substituted with either of the isobaric secondary branched Cl2:0(3‐ O ‐C14:0)) or (14:0(3‐O‐12:0)) fatty acids. Thus, it is unavoidable that during CID‐MS/MS analyses of some isobaric precursor molecular anions, 'similar' product anions having the same m/z values will be obtained . In addition, it will be unavoidable that the CID‐MS/MS fragmentation patterns observed for the isobaric precursor anions will be different.…”

“…It is also imperative to mention that a similar series of GlcN disaccharide and monosaccharide anions was elucidated for the heterogeneous mixtures of lipid A n isolated from another phage-treated LPS SJ-19a. 28 Nevertheless, we would like to stress that the anions obtained for the two series were different, although they had similar m/z values; these compounds were in fact isobars and diastereoisomers. obtained by performing in-source dissociation 31,32 was selected for a quasi-MS 3 analysis (Figure 3, Scheme 4, and Table 3).…”

“…We suggested that the remaining biomolecular members of this heterogonous mixture of lipid A 2‐7 were formed from the incomplete biosynthesis of the respective LPS SJ‐19a. The combined MS/MS studies indicated that all the structures of lipid A 1–5 contained one phosphate group at the O‐4′ position, and were synthesized by a (H 2 PO 4 →4‐O‐)‐β‐D‐GlcpN‐(1→6)‐α‐D‐GlcpN disaccharide blueprint, substituted with (C14:0(3‐OH)) at the N‐2 and O‐3 positions, and contained different substitution permutations of the secondary branched (14:0(3‐O‐12:0)) and (C14:0(3‐O‐14:0)) fatty acids at the N‐2′ and O‐3′ positions …”