(https://mrl.colostate.edu/mtb/) [30] and MycoMass (https://
www.brighamandwomens.org/research/departments/rheumatol
ogy-immunology-allergy/moody-lab/overview)
[ 12],
respectively.
If the M. tuberculosis lipidomic field has made significant progress, it lags behind the lipidomics of eukaryotic lipids because it still
suffers from several shortcomings: the lack of a MS/MS database
that would allow Multiple Reaction Monitoring mode analysis for
the identification of lipids, the poor availability of standards to
achieve absolute quantification, and the lack of a straightforward
bioinformatics pipeline for high-throughput data analysis, especially for unbiased and comparative analysis of large number of
datasets.
Based on the previously published method that relies on diol
chromatography coupled to (ESI)-MS [12], this chapter describes
a lipidomic approach to perform the simultaneous screening of
more than 20 of the main M. tuberculosis lipid families. This
method has been successfully used in the past to characterize
M. tuberculosis lipid profile in response to stress [31], to compare
the lipid profile of mycobacteria strains [32], and also to detect
mycobacterial lipid in tissues [32]. The mass spectrometer available
in the environment of a given laboratory is a limiting and inflexible
factor, given the cost of such an instrument. Thus, the parts of this
chapter describing the analytical detection of mycobacterial lipids
and the data analysis correspond to recommendations to implement this approach and ensure quality of the analyses rather than a
wet-lab protocol.
2 Materials
2.1 Glycolipid
Extraction from
Mycobacteria Culture
1. Chemical fume hood.
2. LC-MS-grade chloroform (CHCl 3 ) or dichloromethane
(CH 2 Cl 2 ) (see Note 1).
3. LC-MS-grade methanol (CH 3 OH).
4. LC-MS-grade water.
5. 1:2 chloroform:methanol (v:v).
6. 1:1 chloroform:methanol (v:v).
7. 2:1 chloroform:methanol (v:v).
8. Internal lipid standard (see Note 5).
9. Solvent evaporation system: centrifugal or parallel evaporator
or nitrogen stream.
10. 50 mL polypropylene V-bottom tubes (Falcon type).
11. 50 mL screw centrifuge glass tubes (Pyrex quality) with PTFE
coated screw caps.
552
Emilie Layre
www.brighamandwomens.org/research/departments/rheumatol
ogy-immunology-allergy/moody-lab/overview)
[ 12],
respectively.
If the M. tuberculosis lipidomic field has made significant progress, it lags behind the lipidomics of eukaryotic lipids because it still
suffers from several shortcomings: the lack of a MS/MS database
that would allow Multiple Reaction Monitoring mode analysis for
the identification of lipids, the poor availability of standards to
achieve absolute quantification, and the lack of a straightforward
bioinformatics pipeline for high-throughput data analysis, especially for unbiased and comparative analysis of large number of
datasets.
Based on the previously published method that relies on diol
chromatography coupled to (ESI)-MS [12], this chapter describes
a lipidomic approach to perform the simultaneous screening of
more than 20 of the main M. tuberculosis lipid families. This
method has been successfully used in the past to characterize
M. tuberculosis lipid profile in response to stress [31], to compare
the lipid profile of mycobacteria strains [32], and also to detect
mycobacterial lipid in tissues [32]. The mass spectrometer available
in the environment of a given laboratory is a limiting and inflexible
factor, given the cost of such an instrument. Thus, the parts of this
chapter describing the analytical detection of mycobacterial lipids
and the data analysis correspond to recommendations to implement this approach and ensure quality of the analyses rather than a
wet-lab protocol.
2 Materials
2.1 Glycolipid
Extraction from
Mycobacteria Culture
1. Chemical fume hood.
2. LC-MS-grade chloroform (CHCl 3 ) or dichloromethane
(CH 2 Cl 2 ) (see Note 1).
3. LC-MS-grade methanol (CH 3 OH).
4. LC-MS-grade water.
5. 1:2 chloroform:methanol (v:v).
6. 1:1 chloroform:methanol (v:v).
7. 2:1 chloroform:methanol (v:v).
8. Internal lipid standard (see Note 5).
9. Solvent evaporation system: centrifugal or parallel evaporator
or nitrogen stream.
10. 50 mL polypropylene V-bottom tubes (Falcon type).
11. 50 mL screw centrifuge glass tubes (Pyrex quality) with PTFE
coated screw caps.
552
Emilie Layre