the target or mechanism of action of new and existing compounds
and enabling direct measurements of their penetration and intracellular biotransformation products, as recently exemplified for
para-aminosalicylic acid, verapamil, bedaquiline, and others
[19–26].
1.2 Experimental
Design
From an analytical perspective, recent advances in chromatography
and mass spectrometry techniques have made it possible to measure
thousands of metabolites with sub-picomolar-level sensitivity and
in the absence of any biological preconception of the analytes of
interest [1, 27]. In this chapter, we describe experimental methodology and analytical approaches to capture and characterize the
Mycobacterium tuberculosis metabolome with minimal samplinginduced perturbation and broad and sensitive recovery of metabolites in their native state. Additional techniques such as the use of
stable isotope labeling for flux analyses in intact M. tuberculosis are
also discussed. Finally, we include a detailed description of an assay
termed activity-based metabolomic profiling (ABMP), used to
identify enzymatic activities of genes of unknown function
[5, 6]. Together, these methods and assays are intended to help
promote the more widespread use of metabolomic technologies in
the context of M. tuberculosis physiology and pathogenesis.
1.3 Filter Culture
System
The short biological half-lives of small metabolites necessitate a
method that enables rapid quenching of samples without prolonged manipulation or exposure to stress. We therefore describe
a filter culture-based system that enables nondisruptive handling,
rapid quenching, and collection of bacterial cells in the absence of
intermediate steps—such as pipetting or centrifugation—that
could introduce sampling-induced changes or losses. The use of
this system further supports dense growth of M. tuberculosis in a
biofilm that preserves its capsule, supports bulk growth at rates
similar to those associated with planktonic liquid cultures, and
enables reproducible detection of most intermediates of central
carbon metabolism (Fig. 1).
1.4 Qualitative vs.
Quantitative
Measurement
Metabolite levels acquired through LC-MS analysis provide a qualitative measure of abundance, allowing for comparison of metabolite levels across samples within a single experiment and providing
relative values of individual intermediates. Typically, metabolite
abundances are normalized to baseline measurements such as protein content or average metabolite ion intensity to facilitate this
comparison. Important limitations of this approach include the fact
that measured values only represent relative, and not absolute,
measures of metabolite abundance due to differences in the intrinsic ionization potentials (and hence response factors) of different
metabolites. Quantitative assessment of metabolite abundance can
only be achieved with the use of external or internal standards
580
Kyle A. Planck and Kyu Rhee
and enabling direct measurements of their penetration and intracellular biotransformation products, as recently exemplified for
para-aminosalicylic acid, verapamil, bedaquiline, and others
[19–26].
1.2 Experimental
Design
From an analytical perspective, recent advances in chromatography
and mass spectrometry techniques have made it possible to measure
thousands of metabolites with sub-picomolar-level sensitivity and
in the absence of any biological preconception of the analytes of
interest [1, 27]. In this chapter, we describe experimental methodology and analytical approaches to capture and characterize the
Mycobacterium tuberculosis metabolome with minimal samplinginduced perturbation and broad and sensitive recovery of metabolites in their native state. Additional techniques such as the use of
stable isotope labeling for flux analyses in intact M. tuberculosis are
also discussed. Finally, we include a detailed description of an assay
termed activity-based metabolomic profiling (ABMP), used to
identify enzymatic activities of genes of unknown function
[5, 6]. Together, these methods and assays are intended to help
promote the more widespread use of metabolomic technologies in
the context of M. tuberculosis physiology and pathogenesis.
1.3 Filter Culture
System
The short biological half-lives of small metabolites necessitate a
method that enables rapid quenching of samples without prolonged manipulation or exposure to stress. We therefore describe
a filter culture-based system that enables nondisruptive handling,
rapid quenching, and collection of bacterial cells in the absence of
intermediate steps—such as pipetting or centrifugation—that
could introduce sampling-induced changes or losses. The use of
this system further supports dense growth of M. tuberculosis in a
biofilm that preserves its capsule, supports bulk growth at rates
similar to those associated with planktonic liquid cultures, and
enables reproducible detection of most intermediates of central
carbon metabolism (Fig. 1).
1.4 Qualitative vs.
Quantitative
Measurement
Metabolite levels acquired through LC-MS analysis provide a qualitative measure of abundance, allowing for comparison of metabolite levels across samples within a single experiment and providing
relative values of individual intermediates. Typically, metabolite
abundances are normalized to baseline measurements such as protein content or average metabolite ion intensity to facilitate this
comparison. Important limitations of this approach include the fact
that measured values only represent relative, and not absolute,
measures of metabolite abundance due to differences in the intrinsic ionization potentials (and hence response factors) of different
metabolites. Quantitative assessment of metabolite abundance can
only be achieved with the use of external or internal standards
580
Kyle A. Planck and Kyu Rhee