compared to 10 pg), is more commonly used. Indeed, the combination of MS and NMR provides structural data of diagnostic
values directly on intact molecules without degradation and consuming minute amount of lipid only. They represent tools of choice
for mutant analyses or species identification.
1.3 Quantitative
Analyses of Lipids
TLC is not only a method of visualization. With the development
of high-performance (HP) TLC, the plate resolution has been
improved, the visualization of lipid spots is more homogeneous,
and the developer systems allow the extraction of quantitative data
from chromatograms with the help of calibration curves.
NMR analysis allows the quantification of molecules when their
specific signals are isolated on the spectra. For example, the integration of the signal resonances of methoxyl groups, cyclopropanes, etc., allows the determination of the proportion of
different kinds of mycolic acids in a mixture.
MALDI-TOF allows the comparison of general profiles, but it
is not the best choice to quantify various kinds of lipids, because the
desorption of molecules is not correlated with quantity. HPLCESI-MS is more adapted to the quantification using calibration
curves. ESI-QTOF-MS method, which gives the general profile
of mycolic acids in the negative mode, allows the quantification of
mycolic acids, based on the pyrolysis fragments at m/z 367 and
395 (C 24 and C 26 acids, respectively) obtained from molecular
peaks. ESI-QTOF-MS-MS allows the quantification of each type
of mycolic acid, based on the pyrolytical cleavage of the molecules
(at m/z 367 and 395), by targeting the molecular ion for a given
MA species.
1.4 The
Stereochemistry of
MycobacterialSpecific Acids
Mycobacteria contain genus- and species-specific lipids exhibiting
chiral centers with defined stereochemistry. Among these are MA
and multimethyl-branched FA found in PDIM, SGL, PGL, or PPT.
In MA (Fig. 1), the stereochemistry of the centers at positions
2 and 3 is conserved in all MA characterized to date and shown to
be 2R and 3R. For the simplest MA, as corynomycolic or nocardomycolic acids, the molecular rotation [M] D is +40
[37]. However, this value varies upon the occurrence of methyl branches in
the mero-chain: the carbon bearing the methyl branch adjacent to
the double bond or to the epoxy ring has R configuration, leading
to a negative contribution to the molecular rotation [38–41],
whereas that bearing the methyl branch adjacent to keto-, methoxy-, hydroxy-, and wax ester-MA has S configuration, in agreement with their biosynthetic filiation [18, 42]. The S,S
stereochemistry of the methyl-methoxy function in natural
methoxy-MA of M. tuberculosis has a real impact on the capacity
of MA to bind CD1b and activate T cells, as demonstrated
recently [43].
Lipid and Lipoarabinomannan
115
values directly on intact molecules without degradation and consuming minute amount of lipid only. They represent tools of choice
for mutant analyses or species identification.
1.3 Quantitative
Analyses of Lipids
TLC is not only a method of visualization. With the development
of high-performance (HP) TLC, the plate resolution has been
improved, the visualization of lipid spots is more homogeneous,
and the developer systems allow the extraction of quantitative data
from chromatograms with the help of calibration curves.
NMR analysis allows the quantification of molecules when their
specific signals are isolated on the spectra. For example, the integration of the signal resonances of methoxyl groups, cyclopropanes, etc., allows the determination of the proportion of
different kinds of mycolic acids in a mixture.
MALDI-TOF allows the comparison of general profiles, but it
is not the best choice to quantify various kinds of lipids, because the
desorption of molecules is not correlated with quantity. HPLCESI-MS is more adapted to the quantification using calibration
curves. ESI-QTOF-MS method, which gives the general profile
of mycolic acids in the negative mode, allows the quantification of
mycolic acids, based on the pyrolysis fragments at m/z 367 and
395 (C 24 and C 26 acids, respectively) obtained from molecular
peaks. ESI-QTOF-MS-MS allows the quantification of each type
of mycolic acid, based on the pyrolytical cleavage of the molecules
(at m/z 367 and 395), by targeting the molecular ion for a given
MA species.
1.4 The
Stereochemistry of
MycobacterialSpecific Acids
Mycobacteria contain genus- and species-specific lipids exhibiting
chiral centers with defined stereochemistry. Among these are MA
and multimethyl-branched FA found in PDIM, SGL, PGL, or PPT.
In MA (Fig. 1), the stereochemistry of the centers at positions
2 and 3 is conserved in all MA characterized to date and shown to
be 2R and 3R. For the simplest MA, as corynomycolic or nocardomycolic acids, the molecular rotation [M] D is +40
[37]. However, this value varies upon the occurrence of methyl branches in
the mero-chain: the carbon bearing the methyl branch adjacent to
the double bond or to the epoxy ring has R configuration, leading
to a negative contribution to the molecular rotation [38–41],
whereas that bearing the methyl branch adjacent to keto-, methoxy-, hydroxy-, and wax ester-MA has S configuration, in agreement with their biosynthetic filiation [18, 42]. The S,S
stereochemistry of the methyl-methoxy function in natural
methoxy-MA of M. tuberculosis has a real impact on the capacity
of MA to bind CD1b and activate T cells, as demonstrated
recently [43].
Lipid and Lipoarabinomannan
115