system with a Varian MonoChrom Diol (dihydroxypropyl
group bonded phase) column (150 mm  2 mm, 3 μm particles of 10 Å porosity) and a Varian MonoChrom Diol guard
column (3 μm  4.6 mm) [12]. Elution profile obtained with
the Varian Diol column is presented in Table 1. This column is
discontinued, but equivalent Diol columns with the same characteristics are available (for example, Agilent or Princeton diol
columns). Using equivalent Diol columns leads to similar order
of elution of M. tuberculosis lipid families but with possible
moderate variation in retention time.
3. Mass spectrometers show variable performance (sensitivity and
dynamic range), and M. tuberculosis lipid families have different
ionization properties. Hence, applying this approach to a different mass spectrometer might require the optimization of the
ionization parameters of the instrument in order to allow the
detection of the different lipid families. For information, this
approach has been later successfully setup using a LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap mass spectrometer
(Thermo) coupled to a Dionex Ultimate 3000 HPLC system
and a Xevo G2 Qtof (WATERS) mass spectrometer
(unpublished).
4. Traces of detergents alter lipidomic analyses by inducing
important contaminant mass spectrometry signals and by suppressing the signal of relevant lipids. It is therefore necessary to
take several precautions with regard to the culture media, the
quality of the solvents, and the appropriate use of plastic and
glass materials:
Culture conditions. Mycobacteria are usually grown in standard Middlebrook 7H9 (Difco) or Sauton medium. It is
recommended to avoid the use of detergent such as Tween,
which is routinely employed to reduce the characteristic aggregation of mycobacteria in liquid media. Indeed, detergents
stick to the bacillus envelope and are recovered during lipid
extraction by organic solvents. When growth monitoring by
optical density measurement is required, it is preferable to use
tyloxapol. Another alternative is to prepare a replicate culture in
the presence of detergent in parallel with detergent-free cultures for each strain, in order to estimate their growth phase
based on optical density.
Material. Material is also a source of contaminations.
Methanol is compatible with plastic consumables, but as soon
as chloroform or dichloromethane is used any contact with
plastic consumables (pipette, tubes, and caps) is strictly forbidden and glass material must be used (pipette, syringe, and tubes
with PTFE caps). In addition, glassware washed with soap such
as in common laboratory laundry is a source of contaminations.
Hence, attention has to be paid to clean the glassware twice
Targeted Lipidomics
571
group bonded phase) column (150 mm  2 mm, 3 μm particles of 10 Å porosity) and a Varian MonoChrom Diol guard
column (3 μm  4.6 mm) [12]. Elution profile obtained with
the Varian Diol column is presented in Table 1. This column is
discontinued, but equivalent Diol columns with the same characteristics are available (for example, Agilent or Princeton diol
columns). Using equivalent Diol columns leads to similar order
of elution of M. tuberculosis lipid families but with possible
moderate variation in retention time.
3. Mass spectrometers show variable performance (sensitivity and
dynamic range), and M. tuberculosis lipid families have different
ionization properties. Hence, applying this approach to a different mass spectrometer might require the optimization of the
ionization parameters of the instrument in order to allow the
detection of the different lipid families. For information, this
approach has been later successfully setup using a LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap mass spectrometer
(Thermo) coupled to a Dionex Ultimate 3000 HPLC system
and a Xevo G2 Qtof (WATERS) mass spectrometer
(unpublished).
4. Traces of detergents alter lipidomic analyses by inducing
important contaminant mass spectrometry signals and by suppressing the signal of relevant lipids. It is therefore necessary to
take several precautions with regard to the culture media, the
quality of the solvents, and the appropriate use of plastic and
glass materials:
Culture conditions. Mycobacteria are usually grown in standard Middlebrook 7H9 (Difco) or Sauton medium. It is
recommended to avoid the use of detergent such as Tween,
which is routinely employed to reduce the characteristic aggregation of mycobacteria in liquid media. Indeed, detergents
stick to the bacillus envelope and are recovered during lipid
extraction by organic solvents. When growth monitoring by
optical density measurement is required, it is preferable to use
tyloxapol. Another alternative is to prepare a replicate culture in
the presence of detergent in parallel with detergent-free cultures for each strain, in order to estimate their growth phase
based on optical density.
Material. Material is also a source of contaminations.
Methanol is compatible with plastic consumables, but as soon
as chloroform or dichloromethane is used any contact with
plastic consumables (pipette, tubes, and caps) is strictly forbidden and glass material must be used (pipette, syringe, and tubes
with PTFE caps). In addition, glassware washed with soap such
as in common laboratory laundry is a source of contaminations.
Hence, attention has to be paid to clean the glassware twice
Targeted Lipidomics
571