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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
Author(s) -
Almostafa Mervt,
Allehyane Bashaeer,
Egli Stefana,
Bottaro Christina,
Fridgen Travis D.,
Banoub Joseph
Publication year - 2016
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7540
Subject(s) - chemistry , lipid a , chromatography , mass spectrometry , tandem mass spectrometry , electrospray ionization , triple quadrupole mass spectrometer , selected reaction monitoring , bacteria , genetics , biology
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‐Glc p N‐(1→6)‐α‐D‐Glc p N 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‐Glc p N‐(1→6)‐α‐D‐Glc p N 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.