Structure Determination of Lipid A with Multiple Glycosylation Sites by Tandem MS of Lithium-Adducted Negative Ions

FLATⁿ is a tandem mass spectrometric technique that can be used to rapidly generate spectral information applicable for structural elucidation of lipids like lipid A from Gram-negative bacterial species from a single bacterial colony. In this study, we extend the scope and capability of FLATⁿ by tandem MS fragmentation of lithium-adducted molecular lipid A anions and fragments (FLAT^n-Li) that provides additional structural and diagnostic data from FLATⁿ samples allowing for the discrimination of terminal phosphate modifications in a variety of pathogenic and environmental species. Using FLAT^n-Li, we elucidated the lipid A structure from several bacterial species, including novel structures from arctic bacterioplankton of the Duganella and Massilia genera that favor 4-amino-4-deoxy-l-arabinopyranose (Ara4N) modification at the 1-phosphate position and that demonstrate double glycosylation with Ara4N at the 1 and 4' phosphate positions simultaneously. The structures characterized in this work demonstrate that some environmental psychrophilic species make extensive use of this structural lipid A modification previously characterized as a pathogenic adaptation and the structural basis of resistance to cationic antimicrobial peptides. This observation extends the role of phosphate modification(s) in environmental species adaptation and suggests that Ara4N modification can functionally replace the positive charge of the phosphoethanolamine modification that is more typically found attached to the 1-phosphate position of modified lipid A.