3. Add four drops of hexamethyldisilazane.
4. Add two drops of trimethylchlorosilane.
5. Vortex.
6. Keep for 15 min at RT.
7. Evaporate to dryness under an air or nitrogen stream at roughly
40
C.
8. Compare the mass values before and after silylation by
MALDI-TOF MS: a shift of 73 atom mass unit (amu) of the
pseudo-molecular ions (M+Na)
+ is observed according to the
existence of hydroxyl.
3.18 Polarimetry
of MA
1. Dissolve pure MA in CHCl 3 .
2. Transfer the solution in the polarimetry tube.
3. Read at a wavelength 589 nm (D ray of sodium).
4. Express the rotation value α as [α] D + or À following the
formula:
[α] D ¼ 100  α/l  ρ, where α is the angle of rotation, D is the
wavelength, l is the length (in dm) of the polarimeter tube,
and ρ the concentration of the solution g/100 mL.
Molecular rotation ([M] D ) is expressed as [α] D Â M/100,
where M is the mass of the compound (see Note 19).
3.19 MALDI/TOF-MS
of Individual Types of
MAME (Determination
of MAME Molecular
Weight)
1. Dissolve MAME sample (Subheading 3.14) in chloroform at a
concentration of approximately 1 mM and directly spot onto
the target plate as 0.5 μL droplets, followed by the addition of
0.5 μL of matrix solution with a 2-μL automatic pipette.
2. Allow to crystallize the sample at room temperature.
3. Run MALDI-TOF/TOF-MS and MS/MS (see Note 20).
Data analysis: Table 1 shows the pseudo-molecular mass values
[M+Na]
+ of structurally characterized MAME, highlighting the
metabolic relationships between the individual types (keto- and
wax-ester; α- and epoxy-; keto-, hydroxy-, and methoxy-) of MA
in terms of considering chain lengths and chemical functions.
The short-chain MAME are not presented in this table, i.e., the
dicarboxylic and the C 60 α
0 -MA. The ω-carboxylic acids result from
the cleavage of the wax-ester function of the wax-MA. Their isolation needs drastic conditions of saponification (see Note 10a)
[52]. The ω-carboxylic acids form a homologue series (M+Na)
+
926, 940, 954, 968, and 982 (C57, C58, C59, C60, C61 as free
mycolic acids). Table 1 shows the pseudo-molecular mass values of
the intact wax-ester at their origin.
The α
0 mycolates found in M. smegmatis gave mass spectrum
signals at m/z 924, 952, 980, and 1008, corresponding to sodium
adducts of mycolic acids homologues series containing a single
double bond with 60, 62, 64, and 66 carbon atoms as free acids.
134
Marie-Antoinette Lane ´ elle et al.
4. Add two drops of trimethylchlorosilane.
5. Vortex.
6. Keep for 15 min at RT.
7. Evaporate to dryness under an air or nitrogen stream at roughly
40
C.
8. Compare the mass values before and after silylation by
MALDI-TOF MS: a shift of 73 atom mass unit (amu) of the
pseudo-molecular ions (M+Na)
+ is observed according to the
existence of hydroxyl.
3.18 Polarimetry
of MA
1. Dissolve pure MA in CHCl 3 .
2. Transfer the solution in the polarimetry tube.
3. Read at a wavelength 589 nm (D ray of sodium).
4. Express the rotation value α as [α] D + or À following the
formula:
[α] D ¼ 100  α/l  ρ, where α is the angle of rotation, D is the
wavelength, l is the length (in dm) of the polarimeter tube,
and ρ the concentration of the solution g/100 mL.
Molecular rotation ([M] D ) is expressed as [α] D Â M/100,
where M is the mass of the compound (see Note 19).
3.19 MALDI/TOF-MS
of Individual Types of
MAME (Determination
of MAME Molecular
Weight)
1. Dissolve MAME sample (Subheading 3.14) in chloroform at a
concentration of approximately 1 mM and directly spot onto
the target plate as 0.5 μL droplets, followed by the addition of
0.5 μL of matrix solution with a 2-μL automatic pipette.
2. Allow to crystallize the sample at room temperature.
3. Run MALDI-TOF/TOF-MS and MS/MS (see Note 20).
Data analysis: Table 1 shows the pseudo-molecular mass values
[M+Na]
+ of structurally characterized MAME, highlighting the
metabolic relationships between the individual types (keto- and
wax-ester; α- and epoxy-; keto-, hydroxy-, and methoxy-) of MA
in terms of considering chain lengths and chemical functions.
The short-chain MAME are not presented in this table, i.e., the
dicarboxylic and the C 60 α
0 -MA. The ω-carboxylic acids result from
the cleavage of the wax-ester function of the wax-MA. Their isolation needs drastic conditions of saponification (see Note 10a)
[52]. The ω-carboxylic acids form a homologue series (M+Na)
+
926, 940, 954, 968, and 982 (C57, C58, C59, C60, C61 as free
mycolic acids). Table 1 shows the pseudo-molecular mass values of
the intact wax-ester at their origin.
The α
0 mycolates found in M. smegmatis gave mass spectrum
signals at m/z 924, 952, 980, and 1008, corresponding to sodium
adducts of mycolic acids homologues series containing a single
double bond with 60, 62, 64, and 66 carbon atoms as free acids.
134
Marie-Antoinette Lane ´ elle et al.