6. Transfer the methanolic bacterial suspension to 50 mL screw
centrifuge glass tubes with PTFE-coated screw caps containing
45 mL of 1:2 chloroform:methanol (v:v) (see Note 6).
7. Allow sterilization overnight (see Note 7).
8. Shake tubes on a rocking shaker for about 1 h.
9. Pellet the bacteria at 2000 Â g for 15 min (or until a translucent
supernatant is obtained), at room temperature.
10. Transfer and dry the supernatant in clean 250 mL-round bottom flask or in 15 mL-glass tubes depending on available
evaporating system.
11. Resuspend the pellets in 45 mL of 1:1 chloroform:methanol (v:
v) and repeat steps 8–10. Pool the organic supernatants.
12. Resuspend the pellets in 45 mL of 2:1 chloroform:methanol (v:
v) and repeat steps 8–10. Pool the organic supernatants.
13. Dry the pooled organic supernatants using a centrifugal or
parallel evaporator or under nitrogen stream in a
chemical hood.
14. Resuspend the dried lipids in 1 mL of 1:1 chloroform:methanol (v:v), transfer to a preweighted 2 mL-amber glass vial with
a Hamilton syringe, and dry under a gentle nitrogen stream
without heat.
15. Rinse with additional 1 mL of 1:1 chloroform:methanol (v:v),
add to the 2 mL-amber glass vial with a Hamilton syringe, and
dry under a gentle nitrogen stream without heat.
16. Weight the dry lipids (see Note 8), resuspend at 2 mg/mL in
1:1 chloroform:methanol (v:v), and store at À80
C if not
analyzed immediately (see Note 9).
3.2 HPLC-MS
Analysis
3.2.1 Setup
of the HPLC-MS System
for Targeted Screening
of Mycobacterial Lipids
1. Equilibrate the HPLC Diol column at room temperature with
Solvent A at 0.15 mL/min for 30 min (see Note 10).
2. Establish the (m/z, RT) map of the lipid families of interest
using your HPLC-MS instrument (see Note 11). Inject purified/synthetic M. tuberculosis lipids (see Note 12) or reference
M. tuberculosis lipid extract (see Note 13) and apply the following gradient at room temperature and at a flow rate of
0.15 mL/min as follows:
Time (min)
Solvents A/B (%)
0.0
100/0
10.0
100/0
17.0
50/50
22.0
50/50
(continued)
554
Emilie Layre
centrifuge glass tubes with PTFE-coated screw caps containing
45 mL of 1:2 chloroform:methanol (v:v) (see Note 6).
7. Allow sterilization overnight (see Note 7).
8. Shake tubes on a rocking shaker for about 1 h.
9. Pellet the bacteria at 2000 Â g for 15 min (or until a translucent
supernatant is obtained), at room temperature.
10. Transfer and dry the supernatant in clean 250 mL-round bottom flask or in 15 mL-glass tubes depending on available
evaporating system.
11. Resuspend the pellets in 45 mL of 1:1 chloroform:methanol (v:
v) and repeat steps 8–10. Pool the organic supernatants.
12. Resuspend the pellets in 45 mL of 2:1 chloroform:methanol (v:
v) and repeat steps 8–10. Pool the organic supernatants.
13. Dry the pooled organic supernatants using a centrifugal or
parallel evaporator or under nitrogen stream in a
chemical hood.
14. Resuspend the dried lipids in 1 mL of 1:1 chloroform:methanol (v:v), transfer to a preweighted 2 mL-amber glass vial with
a Hamilton syringe, and dry under a gentle nitrogen stream
without heat.
15. Rinse with additional 1 mL of 1:1 chloroform:methanol (v:v),
add to the 2 mL-amber glass vial with a Hamilton syringe, and
dry under a gentle nitrogen stream without heat.
16. Weight the dry lipids (see Note 8), resuspend at 2 mg/mL in
1:1 chloroform:methanol (v:v), and store at À80
C if not
analyzed immediately (see Note 9).
3.2 HPLC-MS
Analysis
3.2.1 Setup
of the HPLC-MS System
for Targeted Screening
of Mycobacterial Lipids
1. Equilibrate the HPLC Diol column at room temperature with
Solvent A at 0.15 mL/min for 30 min (see Note 10).
2. Establish the (m/z, RT) map of the lipid families of interest
using your HPLC-MS instrument (see Note 11). Inject purified/synthetic M. tuberculosis lipids (see Note 12) or reference
M. tuberculosis lipid extract (see Note 13) and apply the following gradient at room temperature and at a flow rate of
0.15 mL/min as follows:
Time (min)
Solvents A/B (%)
0.0
100/0
10.0
100/0
17.0
50/50
22.0
50/50
(continued)
554
Emilie Layre