lipid and glycolipid families as illustrated by a few examples in
Fig. 1. Among these surface lipidic components, mycolyl esters
such as trehalose mono- and dimycolates (TMM and TDM) are
found for all mycobacteria, with variation in their mycolic acyl
groups (alpha, methoxy, or keto mycolic acids). In contrast, phthiocerol dimycocerosate (PDIM), phenolic glycolipids (PGL), or sulfoglycolipids (SGL) are strain-specific lipid families. Each of these
families exists as acyl-form variants, which number can reach several
dozens. Finally, the bacillus surface is coated with a detergent-labile
capsule layer essentially made of glycans and, to a lower extent,
lipids and proteins [11]. Hence, similar to eukaryotes, mycobacteria produce a repertoire of lipids that contains hundreds of molecular species [12].
Lipoproteins and lipoglycans, which are amphiphilic macromolecules rather than lipids per se, are studied using specific extraction
and analytical methods [13–16]. Hence, we will focus here on the
so-called “extractible” mycobacterial lipids and glycolipids that are
recovered by exposing mycobacteria pellets to organic solvents.
This includes lipids from the cytoplasm, plasma membrane, and
outer leaflet of the mycomembrane. It is important to note that the
mycolic acids of the mycomembrane’s inner leaflet are not included
in the extractible lipid pool. Indeed, in the absence of saponification
to cleave their linkage with the arabinogalactan, they remain associated with the insoluble material after exposure of mycobacteria
with organic solvents.
The purification and analysis by TLC of many of these lipid
families have been widely described [17] and are also detailed in
previous editions of this protocol book [18]. Mass spectrometrybased detection methods relying on the use of MALDI-Tof or ESI/
APCI-MS instruments have also been used for the analysis of MA
[19, 20], SGL [21–23], DIM [22], PIMs [23–26], PGL [23],
TMM [27], TDM [28], and siderophores (mycobactin and carboxymycobactin families) [29] and might be favored for specific
and high throughput analysis of one of these lipid families. More
recently, lipidomic methods have been developed to analyze the
M. tuberculosis lipid repertoire in a more global way. This effort has
faced the challenges associated with the unique and structurally
diverse mycobacterial lipids, which prevented the direct application
of analytical methods developed for eukaryotic lipids. Two main
studies have described HPLC-(ESI)-Q-Tof approaches that differ
in the reverse phase (C18) [30] versus normal phase (diol) [12]
chromatographic method. While the first one separates lipids based
on their acyl groups (by acyl-form), the second one separates lipids
based on their polar head (by family), but both approaches provide
a wide coverage of M. tuberculosis lipidome. Both studies also
released a theoretical database listing the calculated m/z of adducts
of the published M. tuberculosis lipid families, called Mtb LipidDB
Targeted Lipidomics
551
Fig. 1. Among these surface lipidic components, mycolyl esters
such as trehalose mono- and dimycolates (TMM and TDM) are
found for all mycobacteria, with variation in their mycolic acyl
groups (alpha, methoxy, or keto mycolic acids). In contrast, phthiocerol dimycocerosate (PDIM), phenolic glycolipids (PGL), or sulfoglycolipids (SGL) are strain-specific lipid families. Each of these
families exists as acyl-form variants, which number can reach several
dozens. Finally, the bacillus surface is coated with a detergent-labile
capsule layer essentially made of glycans and, to a lower extent,
lipids and proteins [11]. Hence, similar to eukaryotes, mycobacteria produce a repertoire of lipids that contains hundreds of molecular species [12].
Lipoproteins and lipoglycans, which are amphiphilic macromolecules rather than lipids per se, are studied using specific extraction
and analytical methods [13–16]. Hence, we will focus here on the
so-called “extractible” mycobacterial lipids and glycolipids that are
recovered by exposing mycobacteria pellets to organic solvents.
This includes lipids from the cytoplasm, plasma membrane, and
outer leaflet of the mycomembrane. It is important to note that the
mycolic acids of the mycomembrane’s inner leaflet are not included
in the extractible lipid pool. Indeed, in the absence of saponification
to cleave their linkage with the arabinogalactan, they remain associated with the insoluble material after exposure of mycobacteria
with organic solvents.
The purification and analysis by TLC of many of these lipid
families have been widely described [17] and are also detailed in
previous editions of this protocol book [18]. Mass spectrometrybased detection methods relying on the use of MALDI-Tof or ESI/
APCI-MS instruments have also been used for the analysis of MA
[19, 20], SGL [21–23], DIM [22], PIMs [23–26], PGL [23],
TMM [27], TDM [28], and siderophores (mycobactin and carboxymycobactin families) [29] and might be favored for specific
and high throughput analysis of one of these lipid families. More
recently, lipidomic methods have been developed to analyze the
M. tuberculosis lipid repertoire in a more global way. This effort has
faced the challenges associated with the unique and structurally
diverse mycobacterial lipids, which prevented the direct application
of analytical methods developed for eukaryotic lipids. Two main
studies have described HPLC-(ESI)-Q-Tof approaches that differ
in the reverse phase (C18) [30] versus normal phase (diol) [12]
chromatographic method. While the first one separates lipids based
on their acyl groups (by acyl-form), the second one separates lipids
based on their polar head (by family), but both approaches provide
a wide coverage of M. tuberculosis lipidome. Both studies also
released a theoretical database listing the calculated m/z of adducts
of the published M. tuberculosis lipid families, called Mtb LipidDB
Targeted Lipidomics
551