Premium
A sensitive liquid chromatography/mass spectrometry‐based assay for quantitation of amino‐containing moieties in lipid A
Author(s) -
Kalhorn Thomas F.,
Kiavand Anahita,
Cohen Ilana E.,
Nelson Amanda K.,
Ernst Robert K.
Publication year - 2009
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.3900
Subject(s) - chemistry , lipid a , chromatography , salmonella , biochemistry , neuraminic acid , mass spectrometry , lipopolysaccharide , bacteria , sialic acid , medicine , biology , genetics , endocrinology
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. Copyright © 2009 John Wiley & Sons, Ltd.