Lipoarabinomannans (LAMs) are complex glycoconjugates found in the mycobacterial cell walls (Puzo, 1993; Brennan and Nikaido, 1995). Like the lipopolysaccharides (LPSs) of Gram-negative bacteria, LAMs are amphipatic molecules. Chemically, LAMs consist of a bipartite structure (Puzo, 1993; Brennan and Nikaido 1995) assigned to a polysaccharide core and a hydrophobic anchor referred to as phosphatidyl-myo-inositol. The polysaccharide core can be subdivided into two homopolysaccharides, according to their monosaccharide composition namely, mannan and arabinan. The former consists of a segment of d-Manp-([alpha]->6) with side chains formed by a single [alpha]-d-Manp attached to the C2. Arabinan is composed of a linear chain of d-Araf-([alpha]->5) with oligoarabinofuranosyl side chains branched at the C3. The phosphatidyl-myo-inositol anchor is located at the reducing end of the mannan domain. All the LAMs investigated to date share this bipartite structure, but differ at their reducing ends in the structure and composition of the fatty acids from the phosphatidyl-myo-inositol anchor (Nigou et al., 1997) and in the motifs capping the arabinofuranosyl side chains. These latter motifs have been identified as mannooligosaccharides (Chatterjee et al., 1992; Venisse et al., 1993), and as phospho-myo-inositol leading to the classification of LAMs into lipoarabinomannan with mannosyl capping (ManLAMs) and phosphoinositol-lipo-arabinomannan (PI-LAMs), respectively. In the case of ManLAMs from Mycobacterium bovis BCG, succinyl residues were located on the C2 of 3,5-di-O-linked-[alpha]-Araf residues (Delmas et al., 1997).
ManLAMs have been isolated from different strains of Mycobacterium tuberculosis (Chatterjee et al., 1992), M.bovis BCG (Venisse et al.,1993), and Mycobacterium leprae (Khoo et al., 1995) whereas PI-LAMs were identified in the case of a non-virulent mycobacterial species: Mycobacterium smegmatis (Gilleron et al., 1997).
PI-LAMs and ManLAMs appear to be pivotal mycobacterial antigens. It is now clear that their immunological activities are modulated by both the caps and the anchor. For example, mannooligosaccharide caps and fatty acid residues are required in binding of the ManLAM to the mannose receptor (Schlesinger et al., 1995; Venisse et al., 1995), which mediates the adhesion of virulent mycobacteria to the macrophages, and also in the recognition process of the ManLAMs by the T[alpha][beta] CD4-CD8- cell (Sieling, et al.,1995).
The ManLAMs from M.bovis BCG, M.tuberculosis, and M.leprae are capped by identical mannooligosaccharides composed of one, two, and three Manp unit among which the disaccharide form is the major motif. However, they differ in the mannooligosaccharide capping frequency (Khoo et al., 1995). More recently, in the case of M.bovis BCG, the presence of so-called parietal and cellular ManLAMs has been reported (Nigou et al., 1997). They differ in their mannooligosaccharide capping frequency as well as in the structure of the anchor lipid part (Nigou et al., 1997).
The presence of the mannooligosaccharide caps was directly determined from the intact ManLAMs using nuclear magnetic resonance (NMR) spectrometry 2D 1H-13C heteronuclear experiments (Venisse et al., 1993). However, this approach is limited by the need for purified LAMs in millimolar amounts and the absence of data concerning the mannooligosaccharide structures. Their location, sequence, and number of monosaccharide units were determined by degradative methods involving their release by hydrolysis under mild acidic conditions, fractionation by gel filtration, reducing end tagging by aminobenzoate ethyl ester (ABEE), and purification by HPLC, followed finally by their analysis by fast atom bombardment (FAB/MS) and FAB-MS/MS (Venisse et al., 1993). Their complete structures involving the anomeric configuration and interglycosidic linkage were routinely determined by NMR and alditol acetate analysis.
A new analytical approach using CE coupled with ESI-MS, which combines the high resolving power of CE for the separation of carbohydrates and the structural data provided by the ESI-mass spectra data for identification of the ManLAMs mannooligosaccharide caps, is reported here. This approach includes ManLAMs mild acid hydrolysis followed by oligosaccharide reducing end tagging by APTS that can be carried out without a need for mannooligosaccharide purification. Tagging of mono and oligosaccharides with APTS permits high-resolution CE chromatography and their detection by fluorescence with good sensitivity, and improves their ionization by ESI-MS in negative mode.
Electrophoretic separation of APTS-labeled mono- and oligosaccharides
The polysaccharidic core of the LAMs is exclusively composed of neutral monosaccharides assigned to d-Araf (aldopentose) and [alpha]-d-Manp (aldohexose) which do not significantly absorb in the UV light. A derivatization procedure was therefore applied, and the fluorescent dye, 1-aminopyrene-3,6,8-trisulfonate (APTS) was selected (Evangelista et al., 1995). The optimal excitation of 490 nm for APTS-labeled oligosaccharides matched with the 488 nm of the Ar-ion laser which equipped the CE instrument used (Guttman, 1996). Moreover, this excitation value was close to the fluorescence value of 520 nm allowing a lower limit of detection. Other related fluorescent dyes, such as 8-aminonaphtalene-1,3,6-trisulfonate (ANTS) and disulfonate (ANDS) have also been widely used successfully for oligosaccharide labeling (Chiesa and Horvàth, 1993). However, their fluorescence characteristics are less satisfactory than those of the APTS and require a He-Cd laser (Paulus and Klockow, 1996). All these fluorescent dyes are routinely attached to the reducing termini of each carbohydrate by reductive amination. The fluorophore labeling efficiency was greater than 90%, and no labeling selectivity was observed during derivatization process of the carbohydrate under investigation as had previously been reported by Guttman et al. (1996).
Figure 1. Electropherogram of APTS-labeled standards mono-and oligosaccharides residues (500 fmol each). (A) Triethylammonium phosphate buffer, pH 2.4 (50 mM phosphate, 30 mM TEA). Peak assignment: 1 (arabinose-APTS, 5.78 min); 2 (mannose-APTS, 6.08 min); 3 (maltose-APTS, 6.73 min); 4 (maltotriose-APTS, 7.60 min); 5 (maltotetraose-APTS, 8.58 min). (B) Triethylammonium phosphate buffer pH 1.9 (50 mM phosphate, 10 mM TEA). Peak assignment: 1 (arabinose-APTS, 5.87 min); 2 (mannose-APTS, 6.18 min); 3 (maltose-APTS, 6.93 min); 4 (maltotriose-APTS, 7.82 min); 5 (maltotetraose-APTS, 8.74 min). Conditions: capillary L = 47 cm, hydrodynamic injection 5 sec, temperature 25°C, applied field strength 500 V/cm; detection by LIF (488/520 nm).
The electropherogram in Figure
The amount of TEA needed to titrate phosphoric acid to pH 2.4 (~30 mM) was sufficient to impart a positive charge to the inner wall of the capillary, so that an electroendosmotic flow toward the anodic end was generated when the voltage was applied thus enhancing the speed of analysis. To verify this point, the amount of TEA was decreased from 30 mM to 10 mM, leading to a final pH of 1.9. Under these conditions, the electropherogram depicted in Figure
As depicted in Figure
Capillary zone electrophoresis-electrospray ionization mass spectrometry of APTS-carbohydrate derivatives
The mixture of arabinose, mannose, maltose, maltotriose, and maltotetraose APTS-derivatives was subjected to analysis by CE/ESI-MS in negative mode. The optimized CE separation conditions described above and the triethylammonium phosphate buffer were selected. Under these conditions, the ionization process by electrospray led to an unstable total ionic current (TIC). Moreover, the mass spectra recorded were characterized by a high ionic background and the absence of the expected pseudomolecular ions from the analytes. We postulated, that this phenomenon arose from the incompatibility of the phosphate buffer with the electrospray ionization process. To overcome this problem, other buffers composed of more volatile acids known to be more suitable for ESI /MS were investigated (Johnson and Tomlinson, 1996; Wahl and Smith, 1994). Thus, acetic and formic acids were selected as buffers for the CE separation of APTS-carbohydrate derivatives and the pH was always adjusted using TEA. As depicted in Figure
Figure 2. Electropherogram of APTS-labeled standards, using two buffer systems for on-line coupling with MS. (A) Triethylammonium acetate buffer, pH 3.5(1% v/v acetic acid, 30 mM TEA). Peak assignment: 1 (arabinose-APTS, 5.82 min); 2 (mannose-APTS, 6.04 min); 3 (maltose-APTS, 6.74 min); 4 (maltotriose, 7.56 min); 5 (maltotetraose, 8.32 min). (B) Triethylammonium formate buffer, pH 2.7 (1% v/v formic acid, 30 mM TEA). Peak assignment are the same as in (A): 5.28 min, 5.49 min, 6.09 min, 6.81 min, and 7.47 min, respectively.
Figure
Figure 3. Analysis of the arabinose-APTS. (A) CE/UV profile (254 nm). Conditions CE: capillary L = 80 cm, UV detection (254 nm) at 20 cm, triethylammonium acetate buffer (1% v/v acetic acid, 30 mM TEA). Peak assignment: 1 (APTS, 2.40 min); 2 (Ara-APTS, 2.53 min). (B) TIC profile from CE/ESI-MS. Conditions CE are the same as in (A). Conditions MS: ESI voltage 4 kV, sheath liquid (isopropanol/water 75/25 v/v, 3 µl/min), sheath gas (N2, 30 psi). (C) Negative ESI mass spectrum of arabinose-APTS (peak 2).
The shorter migration time of the arabinose-APTS (2.6 min) observed in the CE-UV detection (Figure
The extracted mass spectrum taken at the crest of peak 2 is presented in Figure
The triethylammonium formate CE buffer (pH 2.7) was also investigated for analysis of the APTS-derivatives of arabinose, mannose, maltose, maltotriose, and maltotetraose. The TIC electropherogram (Figure
The extracted mass spectrum of the peak at 10.05 min assigned to the maltotetraose derivative (Figure
Analysis by CE and CE/ESI-MS of the mannooligosaccharide caps from the lipoarabinomannans of M.bovis BCG
Mannooligosaccharide caps were released by mild acid hydrolysis (0.1N HCl, 30 min, 110°C) from 2 µg of cellular ManLAMs of M.bovis BCG (Nigou et al., 1997). Under these conditions, preferential ManLAMs cleavages occurred in the arabinan domain leading mainly to the formation of Ara, mannooligosaccharide caps and the mannan core. The reaction subproducts were then labeled by APTS, as described in the Material and methods section, and then analyzed by CE using triethylammonium formate buffer. The separation was controlled by LIF and UV detection. Figure
Figure 4. CE/ESI-MS of APTS-carbohydrate derivatives. (A) TIC Electropherogram profile of APTS-labeled standards. CE conditions: triethylammonium formate buffer (1% v/v formic acid, 30 mM TEA), MS conditions are the same as in Figure 3B. (B) Arabinose-APTS negative mass spectrum (peak 1, 8.90 min). (C) Mannose-APTS negative mass spectrum (peak 2, 9.20 min). (D) Maltotetraose-APTS negative mass spectrum (peak 5, 10.05 min).
Table I.
Peak
Migration time (min)
Measured mass
Sequence
Calculated mass (Da)
(M-H)-
(M-2H)2-
1
8.90
590.1
294.5
APTS-Ara
591.01
2
9.20
620.1
309.6
APTS-Man
621.02
3
9.40
782.3
390.5
APTS-Glc-Glc
783.08
4
9.81
944.4
471.6
APTS-Glc-Glc-Glc
945.13
5
10.05
1106
552.9
APTS-Glc-Glc-Glc-Glc
1107.18
Table II.
Peak | Migration time (min) | Measured mass | Deduced sequence | Calculated mass (Da) | |
(M-H)- | (M-2H)2- | ||||
1 | 13.25 | 590.1 | 294.6 | APTS-Ara | 591.01 |
2 | 13.40 | 620.3 | 309.6 | APTS-Man | 621.02 |
3 | 13.60 | 722.6 | 360.3 | APTS-Ara-Ara | 723.06 |
4 | 14.05 | - | 375.6 | APTS-Ara-Man | 753.07 |
5 | 14.60 | 914.1 | 456.6 | APTS-Ara-Man-Man | 915.12 |
6 | 15.01 | 1046.8 | 522.9 | APTS-Ara-Ara-Man-Man | 1047.15 |
Figure 5. Analysis of the mannooligosaccharide caps from the cellular ManLAMs of M.bovis BCG. (A) CE/UV profile (254nm): CE parameters are the same as those described in the Figure 4 caption. (B) TIC profile from CE/ ESI-MS: CE and MS conditions are the same as those described in the legend of Figure 4. (C) Extracted negative mass spectrum of peak 5 (Man-Man-Ara-APTS) from TIC profile obtained in Figure 5B. Conclusion
This study has demonstrated that APTS-derivatized oligo-saccharides may be analyzed by direct coupling of CE with ESI-MS. APTS-oligosaccharides have already been used for sensitive quantitative and qualitative analysis by CE with LIF detection. According to these results, on-line CE/ESI-MS provides a powerful analytical approach for the separation and characterization of unusual oligosaccharides. This, was shown by the structural characterization of the ManLAMs mannooligo-saccharide caps, which became more reliable after analysis of the ESI spectra. This strategy can be routinely applied to 5 nM of LAM so that it may be used in cap analysis of ManLAM obtained from other mycobacterial species, and also in order to screen banks of BCG bovis mutants with truncated ManLAMs. Preliminary studies by on-line CE/ESI on large APTS-oligosaccharide derivatives indicated a loss of resolution and decrease of the ionization process. This problem might be overcome by using a nanoelectrospray system, instead of an electrospray, and an Iontrap mass spectrometer. An alternative approach based on off-line coupling between CE and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF/MS) can also be proposed as it has recently been established that APTS tagging increases oligosaccharide ionization by MALDI-TOF (Suzuki et al., 1997).
Capillary zone electrophoresis with LIF detector
Capillary zone electrophoresis separations were performed on a P/ACE capillary zone electrophoresis system (Beckman Instruments, Inc.) with the cathode on the injection side and the anode on the detection side (reverse polarity). The samples were injected by applying 0.5 psi pressure for 5 s. The separations were monitored on a 470 cm × 50 µm (I.D) uncoated fused-silica capillary column (Sigma, Division Supelco, St. Quentin, France) with a Beckman laser-induced fluorescence (LIF) detection system using a 4 mW argon-ion laser with an excitation wavelength of 488 nm and an emission filter of 520 nm. The temperature of the capillary in the P/ACE instrument was maintained at 25°C. The LIF detection was determined at a capillary length of 40 cm. The electropherograms were acquired and stored on an Dell/Pentium computer using the system Gold software package (Beckman Instruments, Inc.). The capillary was flushed with HCl 0.1N. Separations were carried out using different buffers, prepared from aqueous solutions of phosphoric acid, acetic acid and formic acid at low pH < 4 adjusted with an aqueous solution of TEA.
Capillary zone electrophoresis-electrospray ionization mass spectrometry analysis
All these experiments were carried out using the Beckman CE described above via a floated FinniganMat ESI source to a FinniganMat triple quadrupole instrument (TSQ-700). The CE capillary replaced the sample capillary of the ESI source and the electrospray process was performed from the tip of this capillary. The CE capillary length was extended beyond the outlet of the cartridge for connection to the ESI source. Thus, the most convenient length to use was of 80 cm. In all the experiments, up to 1 cm of polyimide was removed from the spray tip of the capillary. This tip was extended beyond the sheath liquid needle by 0.5 mm and this distance was empirically determined from the ESI spray current using a micrometer head adapter kit for CE/MS (FinniganMat). The outlet end of the capillary was maintained at 4 kV which was the voltage used during the ESI. The voltage allowing electrophoretic separation of the analytes was the difference between the CE (20 kV) and the ESI (4 kV). The sheath liquid (isopropanol/water, 75/25 v/v) was supplied at a flow rate of 3 µl/min and a sheath gas (nitrogen, 30 psi) were delivered coaxially to the CE capillary. In all cases, the sheath liquid should be degassed daily by sonication. During the CE/ESI-MS experiments, the CE current and ESI current spray should be monitored and used diagnostically. Electropherograms of the APTS-carbohydrate derivatives were obtained using the mixture of acetic or formic acid adjusted to low pH with triethylamine. The UV absorption (254nm) was determined at a capillary length of 20 cm. Mass spectra were acquired in the negative mode using dwell times of 2 ms per 1 Da step in full mass scan mode.
Samples preparation
Monosaccharides and glucose oligomers (maltose, maltotriose, maltotetraose) were purchased from Sigma, trisodium APTS from Interchim (Paris). The APTS-derivatized oligosaccharide samples were used directly after derivatization or stored at -20°C. All buffer solutions were filtered through a 0.2 µm pore size filter.
The carbohydrate solutions were dried through the gyrovap. The dried sugars (around 5 nmol) were then labeled as a result of reductive amination by the adding of 0.5 µl of 0.2 M APTS in 15% acetic acid and the same volume of NaBH3CN 1 M in THF. The reaction was incubated for 90 min at 55°C and then diluted with deionized water in order to stop the reaction.
Cellular ManLAMs were extracted and purified as described previously (Nigou et al., 1997). Five nanoMoles of ManLAMs was hydrolyzed (0.1 M HCl, 110°C, for 30 min). The reaction products were mixed with 0.5 µl of 0.2 M APTS in 15% acetic acid and the same volume of NaBH3CN 1 M in THF.
This work was supported by grants from the Région Midi-Pyrénées (RECH/9407528) and from the Mission Scientifique et Technique du Ministère de l'Education Nationale, de l'Enseignement Superieur et de la Recherche (ACC SV6 9506005). We thank FinniganMat (France) and Beckman (France) for technical assistance and valuable discussions regarding approaches to on-line CE/ESI-MS.
ABEE, aminobenzoate ethyl ester; ANDS, 3-amino-naphtalene-2,7-disulfonic acid; ANTS, 8-amino-naphtalene-1,3,6-trisulfonic acid; APTS, 9-aminopyrene-1,4,6-trisulfonate or 1-aminopyrene-3,6,8-trisulfonate; BCG, bacille de Calmette et Guérin; CE, capillary electrophoresis; CE/ESI-MS, capillary electrophoresis/electrospray ionization-mass spectrometry; FAB, fast atom bombardment ionization mode; FAB-MS/MS, fast atom bombardment-tandem mass spectrometry; HPLC, high performance liquid chromatography; LAM, lipoarabinomannan; LIF, laser induced fluorescence detection; MALDI-TOF/MS, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry; ManLAM, lipoarabinomannan with mannosyl capping; NMR, nuclear magnetic resonance; PI-LAM, phospho-inositol lipoarabinomannan; TEA, triethylamine; TIC, total ion current.
1To whom correspondence should be addressed