mass spectrometry (MALDI-TOF MS), by providing accurate
molecular masses of the different classes of MAME, represents an
attractive method by giving pseudo-molecular ion peaks, which
correspond to sodium adducts [20]. Another alternative method
for determining molecular masses of free MA is electrospray ionization (ESI)-MS [21, 22]. However, the requirement of free acids
for ESI-MS may pose a problem when MA are purified as methyl
esters. Finally, an interesting approach for the analysis of lipids is the
combination of HPLC with ESI-MS, which consists of separation
and identification of lipid without any purification and chemical
modification. This method has been successfully applied to monitor
the MA composition in relation to changes in metabolism of the
bacillus in hypoxic dormancy [21]. More recently, ultraperformance liquid chromatography (UPLC)-MS has been applied
to the characterization of mycolic acids. The method, based on the
partition of molecules between a reverse stationary phase liquid at
45
C and a mobile phase, allows the separation of different types of
mycolic acids, according to both their functions and chain lengths,
followed by their characterization by ESI-MS.
Mass spectrometry, which requires only a few μg of sample, has
been also crucial in the structural analysis of complex lipids. For
instance, field-desorption EI-MS has been used to determine the
molecular weight of GPL and to sequence their peptide and saccharide appendages [23]. Fast-atom bombardment (FAB) MS has
also been very informative in this field [24], notably with the
description of a novel sulfated GPL [25]. Structural determination
of GPL has been recently realized by high-resolution multiple-stage
linear ion-trap mass spectrometry ionization [26].
Matrix-assisted laser-desorption/ionization time-of-flight
(MALDI-TOF) MS, using negative mode, has been applied to
PIM molecular weight determination [27]; interestingly, MS/MS
fragmentation from molecular ions obtained by MALDI-TOF or
ESI allows precise determination of the nature of the fatty acids
located on glycerol or on myoinositol and mannose units in the
native PIM [28]. High-resolution multiple-stage linear ion trap MS
with ESI has been successfully applied to determine the structure of
M. tuberculosis SGL [29–31]. Similarly, MALDI-TOF MS has been
useful for the molecular weight determination of PDIM [32] and
PGL [33] as well as trehalose esters, both TMM and TDM [34–
36]. 10 pmol of purified MA is sufficient to obtain resolved mass
spectra pseudo-molecular (M+Na) ions.
In addition to MS studies, which greatly helped these last
decades in the identification of mycolic acids, by providing not
only accurate molecular masses but also structural information,
NMR, another spectrometric method, has proven to be a very
useful analytical tool for identification of specific functions of lipids
(e.g., cis and trans cyclopropanes, double bonds, methyl branch in
MA). This method, which needs more material than MS (1 mg
114
Marie-Antoinette Lane ´ elle et al.
molecular masses of the different classes of MAME, represents an
attractive method by giving pseudo-molecular ion peaks, which
correspond to sodium adducts [20]. Another alternative method
for determining molecular masses of free MA is electrospray ionization (ESI)-MS [21, 22]. However, the requirement of free acids
for ESI-MS may pose a problem when MA are purified as methyl
esters. Finally, an interesting approach for the analysis of lipids is the
combination of HPLC with ESI-MS, which consists of separation
and identification of lipid without any purification and chemical
modification. This method has been successfully applied to monitor
the MA composition in relation to changes in metabolism of the
bacillus in hypoxic dormancy [21]. More recently, ultraperformance liquid chromatography (UPLC)-MS has been applied
to the characterization of mycolic acids. The method, based on the
partition of molecules between a reverse stationary phase liquid at
45
C and a mobile phase, allows the separation of different types of
mycolic acids, according to both their functions and chain lengths,
followed by their characterization by ESI-MS.
Mass spectrometry, which requires only a few μg of sample, has
been also crucial in the structural analysis of complex lipids. For
instance, field-desorption EI-MS has been used to determine the
molecular weight of GPL and to sequence their peptide and saccharide appendages [23]. Fast-atom bombardment (FAB) MS has
also been very informative in this field [24], notably with the
description of a novel sulfated GPL [25]. Structural determination
of GPL has been recently realized by high-resolution multiple-stage
linear ion-trap mass spectrometry ionization [26].
Matrix-assisted laser-desorption/ionization time-of-flight
(MALDI-TOF) MS, using negative mode, has been applied to
PIM molecular weight determination [27]; interestingly, MS/MS
fragmentation from molecular ions obtained by MALDI-TOF or
ESI allows precise determination of the nature of the fatty acids
located on glycerol or on myoinositol and mannose units in the
native PIM [28]. High-resolution multiple-stage linear ion trap MS
with ESI has been successfully applied to determine the structure of
M. tuberculosis SGL [29–31]. Similarly, MALDI-TOF MS has been
useful for the molecular weight determination of PDIM [32] and
PGL [33] as well as trehalose esters, both TMM and TDM [34–
36]. 10 pmol of purified MA is sufficient to obtain resolved mass
spectra pseudo-molecular (M+Na) ions.
In addition to MS studies, which greatly helped these last
decades in the identification of mycolic acids, by providing not
only accurate molecular masses but also structural information,
NMR, another spectrometric method, has proven to be a very
useful analytical tool for identification of specific functions of lipids
(e.g., cis and trans cyclopropanes, double bonds, methyl branch in
MA). This method, which needs more material than MS (1 mg
114
Marie-Antoinette Lane ´ elle et al.