16. Divide the solution into 100 μL aliquots in appropriate tubes
and flash-freeze. Store at À80
C until ready for further
processing.
17. For the enzymatic reactions, set up two tubes each containing
20 mM Tris-HCl pH 7.2 and 100 μL of small molecule. Use
sterile, deionized H 2 O to achieve a final total volume of 980 μL
(see Note 23).
18. Heat-inactivate a small aliquot (30–40 μL) of purified candidate enzyme at 95
C for 5 min then cool on ice for 5 min.
19. Add 20 μL of heat-inactivated purified candidate enzyme to
one experimental tube and 20 μL native enzyme to the other
(see Notes 24 and 25).
20. Incubate at 37
C and remove 100 μL aliquots at 0 (prior to
enzyme addition), 5, 10, 20, 30, 60, 90, and 120 min after
addition of enzyme (see Note 26). Add each aliquot immediately to 300 μL cold acidic acetonitrile and store at 4
C until
ready for further processing.
21. Centrifuge the quenched aliquots at top speed (18,000 Â g)
for 10 min at 4
C in a benchtop microfuge.
22. Carefully transfer 100 μL of the supernatant (without disturbing the pellet) to an LC-MS vial.
23. Cap and store samples at 4
C. Samples are now ready to be
analyzed by either or both of the LC-MS protocols described in
Subheadings 3.5 and 3.6 (see Note 27).
4 Notes
1. Glucose can be replaced with other carbon sources as required.
2. The choice of filter membrane may depend on the experimental
goal. Low binding nylon filters can be used to prevent drugs or
other chemicals from being trapped and maximize exposure of
the bacteria to compounds of interest.
3. Once sufficient biomass is generated, filters can be moved to
different experimental conditions, such as media containing
antibacterial compounds of interest, as required. A convenient
platform for accomplishing this utilizes “swimming pools”:
Sterile 6-well plates affixed to upside-down plastic caps that
contain liquid medium (Fig. 2). This setup permits the bidirectional exchange of nutrients and compounds between the
medium and the bacteria.
4. LC-MS grade water is not recommended due to leaching of
metal ions from glass bottles.
Metabolomics of Mycobacterium tuberculosis
589
and flash-freeze. Store at À80
C until ready for further
processing.
17. For the enzymatic reactions, set up two tubes each containing
20 mM Tris-HCl pH 7.2 and 100 μL of small molecule. Use
sterile, deionized H 2 O to achieve a final total volume of 980 μL
(see Note 23).
18. Heat-inactivate a small aliquot (30–40 μL) of purified candidate enzyme at 95
C for 5 min then cool on ice for 5 min.
19. Add 20 μL of heat-inactivated purified candidate enzyme to
one experimental tube and 20 μL native enzyme to the other
(see Notes 24 and 25).
20. Incubate at 37
C and remove 100 μL aliquots at 0 (prior to
enzyme addition), 5, 10, 20, 30, 60, 90, and 120 min after
addition of enzyme (see Note 26). Add each aliquot immediately to 300 μL cold acidic acetonitrile and store at 4
C until
ready for further processing.
21. Centrifuge the quenched aliquots at top speed (18,000 Â g)
for 10 min at 4
C in a benchtop microfuge.
22. Carefully transfer 100 μL of the supernatant (without disturbing the pellet) to an LC-MS vial.
23. Cap and store samples at 4
C. Samples are now ready to be
analyzed by either or both of the LC-MS protocols described in
Subheadings 3.5 and 3.6 (see Note 27).
4 Notes
1. Glucose can be replaced with other carbon sources as required.
2. The choice of filter membrane may depend on the experimental
goal. Low binding nylon filters can be used to prevent drugs or
other chemicals from being trapped and maximize exposure of
the bacteria to compounds of interest.
3. Once sufficient biomass is generated, filters can be moved to
different experimental conditions, such as media containing
antibacterial compounds of interest, as required. A convenient
platform for accomplishing this utilizes “swimming pools”:
Sterile 6-well plates affixed to upside-down plastic caps that
contain liquid medium (Fig. 2). This setup permits the bidirectional exchange of nutrients and compounds between the
medium and the bacteria.
4. LC-MS grade water is not recommended due to leaching of
metal ions from glass bottles.
Metabolomics of Mycobacterium tuberculosis
589