ester lipids during long periods of storage, methanol can be
replaced by 2-propanol in the solvent mixture.
10. HPLC solvents A and B should be freshly prepared and should
not be stored. If (light) precipitates are observed when adding
the additives to the solvent mixtures, 1 min in a sonic bath can
help the solubilization. The HPLC system should be equipped
with solvent-resistant gaskets and solvent bottles with
integrated air valve to avoid solvent evaporation. Sufficient
amount of solvents should be prepared in order to analyze all
the samples to be compared with the same batch of solvents
and to avoid retention time variation.
11. For future screening of mycobacterial lipids in different samples or for different mycobacteria strains, it is necessary to first
establish a detection map (m/z, retention time) of these different families for a given HPLC-MS system. Mass spectrometers
show variable performance (sensitivity and dynamic range),
and M. tuberculosis glycolipid families have different ionization
properties. Hence, applying this approach with a given mass
spectrometer might require the optimization of the ionization
parameters of the instrument in order to allow the detection of
all lipid families. For information, initially set up using an
Agilent 6520 (ESI)-Q-tof mass spectrometer, this approach
has been later successfully transferred on a Thermo LTQ Orbitrap XL and Waters Xevo G2 Qtof mass spectrometers (unpublished). Using the Agilent Technologies 6520 Accurate-Mass
Q-Tof in full scan mode, recommended Dual ESI source and
acquisition parameters are as follows, as previously published
[1]: gas temperature 325
C, 5 L/min drying gas flow, 30 psig
nebulizer pressure, and VCap 5500 V. Spectra are collected
from m/z 100 to 3000 at 1 spectrum/s with continuous
infusion of calibrants (m/z 121.050873 and 922.009798 in
positive ion mode and m/z 112.985587 and 1033.98810 in
negative ion mode).
12. Standards of mycobacterial lipids are currently poorly marketed. Polar Avanti mycolic acids are distributed by Sigma
Aldrich, and “Mycobacterium tuberculosis, Strain H37Rv,
Total Lipids” (NR-14837) is provided by the BEI resources.
Alternatives for MS signal optimization are to directly work
with the total lipid extract from M. tuberculosis culture to
reproduce the lipid profile detailed in Table 1 or to contact
laboratories with expertise in M. tuberculosis lipid purification
or organic synthesis to obtain pure lipids for the optimization
of individual lipid family detection.
13. The amount of lipids to be injected depends on the instrument
sensibility. Agilent Technologies 6520 Accurate-Mass Q-Tof
requires about 10 micrograms of M. tuberculosis total lipid
extract to detect the different lipid families listed in Table 1.
Targeted Lipidomics
573
replaced by 2-propanol in the solvent mixture.
10. HPLC solvents A and B should be freshly prepared and should
not be stored. If (light) precipitates are observed when adding
the additives to the solvent mixtures, 1 min in a sonic bath can
help the solubilization. The HPLC system should be equipped
with solvent-resistant gaskets and solvent bottles with
integrated air valve to avoid solvent evaporation. Sufficient
amount of solvents should be prepared in order to analyze all
the samples to be compared with the same batch of solvents
and to avoid retention time variation.
11. For future screening of mycobacterial lipids in different samples or for different mycobacteria strains, it is necessary to first
establish a detection map (m/z, retention time) of these different families for a given HPLC-MS system. Mass spectrometers
show variable performance (sensitivity and dynamic range),
and M. tuberculosis glycolipid families have different ionization
properties. Hence, applying this approach with a given mass
spectrometer might require the optimization of the ionization
parameters of the instrument in order to allow the detection of
all lipid families. For information, initially set up using an
Agilent 6520 (ESI)-Q-tof mass spectrometer, this approach
has been later successfully transferred on a Thermo LTQ Orbitrap XL and Waters Xevo G2 Qtof mass spectrometers (unpublished). Using the Agilent Technologies 6520 Accurate-Mass
Q-Tof in full scan mode, recommended Dual ESI source and
acquisition parameters are as follows, as previously published
[1]: gas temperature 325
C, 5 L/min drying gas flow, 30 psig
nebulizer pressure, and VCap 5500 V. Spectra are collected
from m/z 100 to 3000 at 1 spectrum/s with continuous
infusion of calibrants (m/z 121.050873 and 922.009798 in
positive ion mode and m/z 112.985587 and 1033.98810 in
negative ion mode).
12. Standards of mycobacterial lipids are currently poorly marketed. Polar Avanti mycolic acids are distributed by Sigma
Aldrich, and “Mycobacterium tuberculosis, Strain H37Rv,
Total Lipids” (NR-14837) is provided by the BEI resources.
Alternatives for MS signal optimization are to directly work
with the total lipid extract from M. tuberculosis culture to
reproduce the lipid profile detailed in Table 1 or to contact
laboratories with expertise in M. tuberculosis lipid purification
or organic synthesis to obtain pure lipids for the optimization
of individual lipid family detection.
13. The amount of lipids to be injected depends on the instrument
sensibility. Agilent Technologies 6520 Accurate-Mass Q-Tof
requires about 10 micrograms of M. tuberculosis total lipid
extract to detect the different lipid families listed in Table 1.
Targeted Lipidomics
573