3.7 Activity-Based
Metabolomic Profiling
(ABMP) Sample
Preparation (See
Note 15)
1. Inoculate 100 mL pre-warmed 7H9-ADN medium with a
single colony or freezer stock of Mycobacterium bovis BCG
(see Notes 16 and 17).
2. Incubate (rolling or shaking) at 37
C until the optical density
at 600 nm (OD 600 ) reaches 1.0 (see Note 18).
3. Add the starter culture to 900 mL pre-warmed 7H9-ADN
medium (see Note 19).
4. Incubate (rolling or shaking) at 37
C until the OD 600 reaches
1.0 (about 3 days).
5. Cool cells on ice for 10–15 min.
6. Transfer to centrifuge buckets and centrifuge at 7000 Â g for
15 min at 4
C.
7. Discard the supernatant and resuspend the cells in an equal
volume of ice-cold 7H9-ADN medium.
8. Repeat steps 6 and 7 once (see Note 20).
9. Harvest the cells (as in step 6), discard supernatant, and store
on ice (see Note 21).
10. Add 1 volume cold metabolite extraction buffer to the cell
pellet.
11. Disrupt the cells by probe sonication on ice for six 30-s
intervals.
12. Separate soluble and insoluble fractions by centrifugation at
27,000 Â g for 1 h at 4
C.
13. Carefully remove the soluble fraction (without disturbing the
pellet) and transfer to a new container on ice. Discard the
insoluble debris.
14. Flash-freeze the soluble material and freeze-dry.
15. Suspend the resulting powder in 4 mL cold 20 mM Tris-HCl
pH 7.2 to generate small-molecule extracts (see Note 22).
Table 2
Ion-pairing reversed-phase LC-MS gradient
Time (min)
Gradient (% solvent B)
0–3.5
0
4–7.5
30
8–15
35
20–24
99
24–24.5
0
588
Kyle A. Planck and Kyu Rhee
Metabolomic Profiling
(ABMP) Sample
Preparation (See
Note 15)
1. Inoculate 100 mL pre-warmed 7H9-ADN medium with a
single colony or freezer stock of Mycobacterium bovis BCG
(see Notes 16 and 17).
2. Incubate (rolling or shaking) at 37
C until the optical density
at 600 nm (OD 600 ) reaches 1.0 (see Note 18).
3. Add the starter culture to 900 mL pre-warmed 7H9-ADN
medium (see Note 19).
4. Incubate (rolling or shaking) at 37
C until the OD 600 reaches
1.0 (about 3 days).
5. Cool cells on ice for 10–15 min.
6. Transfer to centrifuge buckets and centrifuge at 7000 Â g for
15 min at 4
C.
7. Discard the supernatant and resuspend the cells in an equal
volume of ice-cold 7H9-ADN medium.
8. Repeat steps 6 and 7 once (see Note 20).
9. Harvest the cells (as in step 6), discard supernatant, and store
on ice (see Note 21).
10. Add 1 volume cold metabolite extraction buffer to the cell
pellet.
11. Disrupt the cells by probe sonication on ice for six 30-s
intervals.
12. Separate soluble and insoluble fractions by centrifugation at
27,000 Â g for 1 h at 4
C.
13. Carefully remove the soluble fraction (without disturbing the
pellet) and transfer to a new container on ice. Discard the
insoluble debris.
14. Flash-freeze the soluble material and freeze-dry.
15. Suspend the resulting powder in 4 mL cold 20 mM Tris-HCl
pH 7.2 to generate small-molecule extracts (see Note 22).
Table 2
Ion-pairing reversed-phase LC-MS gradient
Time (min)
Gradient (% solvent B)
0–3.5
0
4–7.5
30
8–15
35
20–24
99
24–24.5
0
588
Kyle A. Planck and Kyu Rhee