5. Higher cell densities may be used for mutant strains that are
difficult to grow on plates.
6. Larger volumes of culture can be deposited on the filters, to
insure adequate biomass for LC-MS analysis at the end of the
experiment.
7. Bacteria are ideally collected at mid-log phase growth on filters.
This is typically achieved after 5–7 days of filter growth on
7H10 plates with glucose and glycerol as carbon sources. The
experimental design can be altered to insure that this growth
phase is achieved at sample collection when alternate media or
strains/mutants are used. Shorter or longer growth intervals
can also be used to observe growth phase-specific differences in
metabolism.
8. The cold solution rapidly quenches bacterial metabolism, and
the direct use of collection buffer eliminates steps such as
washing and spinning that could stress the sample.
9. If a cooling system for the beat beater is not available, samples
can be cooled on ice for 30 s between runs.
10. At this point, tubes containing sterile samples can be disinfected and removed from the BSL-3 facility for LC-MS
analysis.
11. The standard method, when run in negative and positive,
suffices for detection and quantitation of most intermediary
metabolites. However, we have developed the ion-pairing
method for improved detection and resolution of highly negatively charged metabolites. Depending on the goal of the
experiment, either or both methods can be used.
12. LC-MS equipment can be obtained from companies such as
Agilent Technologies, the Waters Corporation, the Bruker
Corporation, and the Shimadzu Corporation.
13. Samples should be run in both positive and negative detection
modes.
14. Samples should be run in negative detection mode only.
15. Activity-based metabolomic profiling (ABMP) is not a followon to the protocols described here, but an example of an
application of these methods. ABMP allows for the functional
characterization of orphan enzyme activities through the use of
recombinant enzymes in whole-cell lysates. An unbiased metabolomic approach is used to discover catalytic activities
through the identification of putative physiological products
and substrates.
16. 100 mL is stated as this is the maximum volume permitted in
the 1-L roller bottles employed in our laboratory to routinely
culture M. bovis BCG and M. tuberculosis H37Rv.
590
Kyle A. Planck and Kyu Rhee
difficult to grow on plates.
6. Larger volumes of culture can be deposited on the filters, to
insure adequate biomass for LC-MS analysis at the end of the
experiment.
7. Bacteria are ideally collected at mid-log phase growth on filters.
This is typically achieved after 5–7 days of filter growth on
7H10 plates with glucose and glycerol as carbon sources. The
experimental design can be altered to insure that this growth
phase is achieved at sample collection when alternate media or
strains/mutants are used. Shorter or longer growth intervals
can also be used to observe growth phase-specific differences in
metabolism.
8. The cold solution rapidly quenches bacterial metabolism, and
the direct use of collection buffer eliminates steps such as
washing and spinning that could stress the sample.
9. If a cooling system for the beat beater is not available, samples
can be cooled on ice for 30 s between runs.
10. At this point, tubes containing sterile samples can be disinfected and removed from the BSL-3 facility for LC-MS
analysis.
11. The standard method, when run in negative and positive,
suffices for detection and quantitation of most intermediary
metabolites. However, we have developed the ion-pairing
method for improved detection and resolution of highly negatively charged metabolites. Depending on the goal of the
experiment, either or both methods can be used.
12. LC-MS equipment can be obtained from companies such as
Agilent Technologies, the Waters Corporation, the Bruker
Corporation, and the Shimadzu Corporation.
13. Samples should be run in both positive and negative detection
modes.
14. Samples should be run in negative detection mode only.
15. Activity-based metabolomic profiling (ABMP) is not a followon to the protocols described here, but an example of an
application of these methods. ABMP allows for the functional
characterization of orphan enzyme activities through the use of
recombinant enzymes in whole-cell lysates. An unbiased metabolomic approach is used to discover catalytic activities
through the identification of putative physiological products
and substrates.
16. 100 mL is stated as this is the maximum volume permitted in
the 1-L roller bottles employed in our laboratory to routinely
culture M. bovis BCG and M. tuberculosis H37Rv.
590
Kyle A. Planck and Kyu Rhee