12. 250 mL round bottom flasks if using rotary vacuum evaporator
as drying system or 15 mL-centrifuge glass tubes (Pyrex quality) if using stream-drying system to dry organic phases.
13. 500 μL Hamilton syringes.
14. Pasteur glass pipettes.
15. Rocking shaker.
16. Nitrogen stream drying system.
17. Microbalance.
18. 2 mL-amber vials with PTFE screw caps.
2.2 HPLC-MS
Analysis
1. Mycobacterial lipid standards (see Note 12).
2. LC/MS-grade hexane, 2-isopropanol, methanol, formic acid
(CH 3 COOH), and ammonium hydroxide (NH 4 OH).
3. 1:1 chloroform:methanol (v:v).
4. Solvent A: 70:30 hexane:isopropanol (v:v), 0.1% (v/v) formic
acid, and 0.05% (v/v) Ammonium hydroxide.
5. Solvent B: 30:70 methanol:isopropanol, 0.1% (v/v) formic
acid, and 0.05% (v/v) Ammonium hydroxide.
6. HPLC Diol (dihydroxypropyl group bonded phase) column
(150 mm  2 mm, 3 μm particles of 10 Å porosity) and a Diol
guard column (3 μm  4.6 mm) (see Note 2).
7. High-resolution mass spectrometer (see Note 3).
2.3 Targeted
Screening
and Comparative
Analysis
1. R software.
2. msConvert ProteoWizard.
3. MS database: Mtb LipidDB [30] (https://mrl.colostate.edu/
mtb/) or MycoMass [12] (https://www.brighamandwomens.
org/research/departments/rheumatology-immunologyallergy/moody-lab/overview).
3 Methods
3.1 Glycolipid
Extraction from
Mycobacteria Culture
1. Transfer 50 mL of mycobacterial culture grown in the absence
of detergent (see Note 4) into 50 mL polypropylene V-bottom
tubes.
2. Centrifuge the tubes at 2000 Â g for 15 min at room temperature and discard the supernatant.
3. Wash the pellet twice with 10 mL of water by resuspending the
bacteria and repeating step 2.
4. Resuspend the bacterial pellet in 1 mL of methanol.
5. Add internal lipid standard (see Note 5).
Targeted Lipidomics
553
as drying system or 15 mL-centrifuge glass tubes (Pyrex quality) if using stream-drying system to dry organic phases.
13. 500 μL Hamilton syringes.
14. Pasteur glass pipettes.
15. Rocking shaker.
16. Nitrogen stream drying system.
17. Microbalance.
18. 2 mL-amber vials with PTFE screw caps.
2.2 HPLC-MS
Analysis
1. Mycobacterial lipid standards (see Note 12).
2. LC/MS-grade hexane, 2-isopropanol, methanol, formic acid
(CH 3 COOH), and ammonium hydroxide (NH 4 OH).
3. 1:1 chloroform:methanol (v:v).
4. Solvent A: 70:30 hexane:isopropanol (v:v), 0.1% (v/v) formic
acid, and 0.05% (v/v) Ammonium hydroxide.
5. Solvent B: 30:70 methanol:isopropanol, 0.1% (v/v) formic
acid, and 0.05% (v/v) Ammonium hydroxide.
6. HPLC Diol (dihydroxypropyl group bonded phase) column
(150 mm  2 mm, 3 μm particles of 10 Å porosity) and a Diol
guard column (3 μm  4.6 mm) (see Note 2).
7. High-resolution mass spectrometer (see Note 3).
2.3 Targeted
Screening
and Comparative
Analysis
1. R software.
2. msConvert ProteoWizard.
3. MS database: Mtb LipidDB [30] (https://mrl.colostate.edu/
mtb/) or MycoMass [12] (https://www.brighamandwomens.
org/research/departments/rheumatology-immunologyallergy/moody-lab/overview).
3 Methods
3.1 Glycolipid
Extraction from
Mycobacteria Culture
1. Transfer 50 mL of mycobacterial culture grown in the absence
of detergent (see Note 4) into 50 mL polypropylene V-bottom
tubes.
2. Centrifuge the tubes at 2000 Â g for 15 min at room temperature and discard the supernatant.
3. Wash the pellet twice with 10 mL of water by resuspending the
bacteria and repeating step 2.
4. Resuspend the bacterial pellet in 1 mL of methanol.
5. Add internal lipid standard (see Note 5).
Targeted Lipidomics
553