Development
of Isotope Labeling Liquid Chromatography–Mass
Spectrometry for Metabolic Profiling of Bacterial Cells and Its Application
for Bacterial Differentiation
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Abstract
Quantitative and comprehensive profiling
of cellular metabolites
is currently a challenging task in bacterial metabolomics. In this
work, a simple and robust method for profiling the amine- and phenol-containing
metabolome of bacterial cells is described. The overall workflow consists
of methanol-based cell lysis and metabolite extraction with ultrasonication,
differential isotope dansylation labeling of cellular metabolites,
and analysis of the labeled metabolites by liquid chromatography–mass
spectrometry (LC–MS). Over a thousand peak pairs or putative
metabolites can be detected from bacterial cells in a 25 min LC–MS
run and near 2500 putative metabolites can be found in one bacterium
from combined results of multiple analyses. After careful examination
and optimization of the sample preparation process, this method is
shown to be effective for both Gram-positive and Gram-negative bacteria.
An idea of applying LC–ultraviolet (UV) detection to quantify
the total amount of labeled metabolites is shown to be effective for
normalizing the amounts of metabolites present in different samples
for metabolome comparison. The use of differential isotopic labeling
allows relative quantification of each individual metabolite, which
facilitates comparative metabolomics studies and the generation of
a metabolic fingerprint of a bacterium. Finally, this method is demonstrated
to be useful for the differentiation of three bacterial species in
cultured media and spiked human urine samples