and/or accompanying standard curves for molecules of interest. A
standard curve allows for determination of the linear range of
detection for a particular metabolite and accurate quantification
of the concentrations of individual metabolites within each sample.
1.5 Metabolite Pool
Size Analysis
The analytical approach described herein measures total pools of
small-molecule metabolites at a single point in time, providing a
static picture of cells’ metabolic states. This tool is useful for comparing the impact of different conditions, mutations, and strains on
overall cell metabolism but cannot necessarily be taken as a steadystate representation of cellular physiology. Multiple time points can
provide snapshot views of changes in metabolite levels over time.
1.6 Activity-Based
Metabolomic Profiling
(ABMP)
Our metabolomics method lends itself to several applications for
studying the physiological responses of M. tuberculosis. One example is activity-based metabolomic profiling (ABMP), an assay specifically developed to identify enzymatic activities for genes of
unknown function by leveraging the analytical discriminatory
power of liquid chromatography-coupled high-resolution mass
spectrometry (LC-MS) to analyze the impact of a recombinant
enzyme and potential cofactors on a highly concentrated smallmolecule extract derived from the homologous organism. By monitoring for the matched time and enzyme-dependent depletion and
accumulation of putative substrates and products, this assay enables
the discovery of catalytic, rather than simple binding, activity using
Fig. 1 Experimental design of metabolomics in M. tuberculosis. 1 mL of M. tuberculosis grown to an optical
density (OD 600 ) of 1 is deposited on sterile filters using vacuum filtration. These filters are transferred to 7H10
plates and incubated for 5 days to generate sufficient biomass. Bacteria-laden filters are then transferred onto
liquid medium in “swimming pool” plates for the experimental period, usually 24 h. Samples are then
collected and extracted for analysis by LC-MS as described
Metabolomics of Mycobacterium tuberculosis
581
standard curve allows for determination of the linear range of
detection for a particular metabolite and accurate quantification
of the concentrations of individual metabolites within each sample.
1.5 Metabolite Pool
Size Analysis
The analytical approach described herein measures total pools of
small-molecule metabolites at a single point in time, providing a
static picture of cells’ metabolic states. This tool is useful for comparing the impact of different conditions, mutations, and strains on
overall cell metabolism but cannot necessarily be taken as a steadystate representation of cellular physiology. Multiple time points can
provide snapshot views of changes in metabolite levels over time.
1.6 Activity-Based
Metabolomic Profiling
(ABMP)
Our metabolomics method lends itself to several applications for
studying the physiological responses of M. tuberculosis. One example is activity-based metabolomic profiling (ABMP), an assay specifically developed to identify enzymatic activities for genes of
unknown function by leveraging the analytical discriminatory
power of liquid chromatography-coupled high-resolution mass
spectrometry (LC-MS) to analyze the impact of a recombinant
enzyme and potential cofactors on a highly concentrated smallmolecule extract derived from the homologous organism. By monitoring for the matched time and enzyme-dependent depletion and
accumulation of putative substrates and products, this assay enables
the discovery of catalytic, rather than simple binding, activity using
Fig. 1 Experimental design of metabolomics in M. tuberculosis. 1 mL of M. tuberculosis grown to an optical
density (OD 600 ) of 1 is deposited on sterile filters using vacuum filtration. These filters are transferred to 7H10
plates and incubated for 5 days to generate sufficient biomass. Bacteria-laden filters are then transferred onto
liquid medium in “swimming pool” plates for the experimental period, usually 24 h. Samples are then
collected and extracted for analysis by LC-MS as described
Metabolomics of Mycobacterium tuberculosis
581