17. BCG is used instead of M. tuberculosis to allow processing of
samples outside of a biosafety level 3 environment.
18. Monitor OD 600 every 2 days to begin with. It can take from
5 to 10 days for the culture to reach the correct density.
19. We divide the culture into ten 1-L roller bottles.
20. Ideally the final harvest should result in a single cell pellet.
To achieve this, reduce the amount of wash solution used to
resuspend the cells and pool samples together prior to
centrifugation.
21. For long-term storage cell pellets can be flash-frozen (EtOH:
dry ice bath) and kept at À80
C.
22. This volume generates a 10Â extract for the conditions used in
our laboratory. The buffer composition and volume used are
flexible; adjust accordingly as required for the final application.
23. Add appropriate concentrations of any cofactors (e.g., pyridoxal phosphate, NAD(P)
+ /H, Zn
2+ ) that are predicted to
be required for enzyme activity to both tubes.
24. Final concentration of enzyme in the reaction mixture will vary
depending on the enzyme preparation used. Final concentrations employed in our laboratory typically range from 0.5 to
5 μM.
25. If enzyme quantity is limited, heat-inactivated enzyme can be
substituted with an equal volume of enzyme storage buffer.
26. If precipitation becomes visible, adjustments to the concentration of buffer, enzyme, cofactors, and small-molecule extract
must be made.
27. All metabolite readouts should be normalized to residual protein content (determined by a colorimetric assay) to account
for differences in sample yield and bead-beating efficiency.
Acknowledgment
Authors M. Nandakumar and LPS de Carvalho contributed to the
previous version of this chapter.
References
1. Patti GJ, Yanes O, Siuzdak G (2012) Innovation: metabolomics: the apogee of the omics
trilogy. Nat Rev Mol Cell Biol 13(4):263–269
2. Reaves ML, Rabinowitz JD (2011) Metabolomics in systems microbiology. Curr Opin Biotechnol 22(1):17–25
3. Rhee KY, Carvalho LP, Bryk R, Ehrt S,
Marrero J, Park SW, Schnappinger D,
Venugopal A, Nathan C (2011) Central carbon
metabolism in mycobacterium tuberculosis: an
unexpected frontier. Trends Microbiol
7:307–314
4. Saghatelian A, Cravatt BF (2005) Global strategies to integrate the proteome and metabolome. Curr Opin Chem Biol 9(1):62–68
Metabolomics of Mycobacterium tuberculosis
591
samples outside of a biosafety level 3 environment.
18. Monitor OD 600 every 2 days to begin with. It can take from
5 to 10 days for the culture to reach the correct density.
19. We divide the culture into ten 1-L roller bottles.
20. Ideally the final harvest should result in a single cell pellet.
To achieve this, reduce the amount of wash solution used to
resuspend the cells and pool samples together prior to
centrifugation.
21. For long-term storage cell pellets can be flash-frozen (EtOH:
dry ice bath) and kept at À80
C.
22. This volume generates a 10Â extract for the conditions used in
our laboratory. The buffer composition and volume used are
flexible; adjust accordingly as required for the final application.
23. Add appropriate concentrations of any cofactors (e.g., pyridoxal phosphate, NAD(P)
+ /H, Zn
2+ ) that are predicted to
be required for enzyme activity to both tubes.
24. Final concentration of enzyme in the reaction mixture will vary
depending on the enzyme preparation used. Final concentrations employed in our laboratory typically range from 0.5 to
5 μM.
25. If enzyme quantity is limited, heat-inactivated enzyme can be
substituted with an equal volume of enzyme storage buffer.
26. If precipitation becomes visible, adjustments to the concentration of buffer, enzyme, cofactors, and small-molecule extract
must be made.
27. All metabolite readouts should be normalized to residual protein content (determined by a colorimetric assay) to account
for differences in sample yield and bead-beating efficiency.
Acknowledgment
Authors M. Nandakumar and LPS de Carvalho contributed to the
previous version of this chapter.
References
1. Patti GJ, Yanes O, Siuzdak G (2012) Innovation: metabolomics: the apogee of the omics
trilogy. Nat Rev Mol Cell Biol 13(4):263–269
2. Reaves ML, Rabinowitz JD (2011) Metabolomics in systems microbiology. Curr Opin Biotechnol 22(1):17–25
3. Rhee KY, Carvalho LP, Bryk R, Ehrt S,
Marrero J, Park SW, Schnappinger D,
Venugopal A, Nathan C (2011) Central carbon
metabolism in mycobacterium tuberculosis: an
unexpected frontier. Trends Microbiol
7:307–314
4. Saghatelian A, Cravatt BF (2005) Global strategies to integrate the proteome and metabolome. Curr Opin Chem Biol 9(1):62–68
Metabolomics of Mycobacterium tuberculosis
591