Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony.
The lipid A of Gram-negative bacteria plays a major role in the pathogenesis of bacterial infections. Lipid A diversity is observed both in the number and length of fatty-acid side chains and in the presence of terminal phosphate residues and associated modifications. In this report, we describe a new sample preparation method based on microwave-assisted enzymatic digestion and detergent-free mild hydrolysis, in conjunction with a MALDI-time-of-flight (TOF)/TOF analysis, to determine the structures of lipid A from Helicobacter pylori. The total time for sample preparation and mass spectrometric analysis is within 2 h and applicable to profiling the lipid A structures from dried bacterial cells on as little as 1 microg. The reliability of the technique was further demonstrated through the analysis of the lipid A from bacterial cells of different H. pylori strains. The phosphorylation and acylation patterns of lipid A could be elucidated using material from a single colony. Furthermore, we found unusual heptaacyl lipid A species present in H. pylori mutant that have not been previously reported, although the abundance was relatively low. The present study provides the first characterization of the lipid A component from a single bacterial colony sample by mass spectrometry.
Zhou
Ping
P
Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada.
Chandan
Vandana
V
Liu
Xin
X
Chan
Kenneth
K
Altman
Eleonora
E
Li
Jianjun
J
D053000:Analytic Sample Preparation Methods; D015169:Colony Count, Microbial; D019286:Endopeptidase K / Q000378:metabolism; D016480:Helicobacter pylori / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D006868:Hydrolysis; D008050:Lipid A / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008872:Microwaves; D019346:Sodium Acetate / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D008050:Lipid A; D008070:Lipopolysaccharides; D019346:Sodium Acetate; D019286:Endopeptidase K