A sensitive liquid chromatography/mass spectrometry‐based assay for quantitation of amino‐containing moieties in lipid A

Abstract

A novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, GlcN), 2-amino-2-deoxygalactose (galactosamine, GalN), and 4-amino-4-deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed-phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10-fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium-limited versus magnesium-replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium-replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino-containing moieties in lipid A from a variety of bacterial sources.

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of Acinetobacter baumannii

“…This modification is also seen on the lipid A from Francisella novicida as published previously by our laboratory (25,27) and others in the Francisella field (42,43). In the setting of F. novicida, the enzyme FlmK adds a GalN and a mannose residue to the 1 and 4= positions of lipid A whereas the FlmF2 enzyme is responsible for the addition of the GalN residue.…”

“…In order to confirm the identity of the hexosamine sugar modification to A. baumannii lipid A, we utilized an LC-MS/MS platform previously developed in our laboratory for detection and quantitation of amino sugars isolated from lipid A in Francisella novicida (25)(26)(27). This study demonstrated the detection of the GalN modification to lipid A.…”

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

“…B). Interestingly, this shift corresponds to the molecular weight of galactosamine, and wild‐type Francisella free lipid A is modified with a galactosamine at the 1 position phosphate (Phillips et al ., ; Wang et al ., ; ; Schilling et al ., ; Shaffer et al ., ; Kanistanon et al ., ; Kalhorn et al ., ; Song et al ., ; Soni et al ., ; Beasley et al ., ). The absence of the galactosamine moiety on the free lipid A of the mutant would result in an exposed, negatively charged phosphate group, which correlates with the decreased surface charge of the mutant strain (Fig.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2009–2010

“…The derivatives were separated using RP-chromatography and analyzed with a quadrupole MS to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species (Kalhorn et al, 2009). Mass Spectrometry Reviews DOI 10.1002/mas (Continued) c. Core oligosaccharide During an analysis of the permethylated derivative of the core oligosaccharide from Aeromonas hydrophila, prepared by the Hakamori method, ions appearing at 78 mass units higher than the [MþH] þ ions were observed.…”