Structural Characterization of Mycobacterium tuberculosis

Apr 10, 2001 - Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) associated with capillary electrophoresis (...
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Anal. Chem. 2001, 73, 2323-2330

Structural Characterization of Mycobacterium tuberculosis Lipoarabinomannans by the Combination of Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Pascal Ludwiczak, The´re`se Brando, Bernard Monsarrat, and Germain Puzo*

Institut de Pharmacologie et de Biologie Structurale, UMR 5089, CNRS, 205 route de Narbonne, 31077 Toulouse cedex, France

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) associated with capillary electrophoresis (CE) has been used for structural characterization of mannooligosaccharide caps from Mycobacterium tuberculosis H37rv mannosylated lipoarabinomannans (ManLAMs). The mannooligosaccharide caps were released by mild acid hydrolysis, labeled with 1-aminopyrene-3,6,8-trisulfonate (APTS) prior to being separated by CE, collected, and analyzed by MALDI-TOFMS and post-source decay experiments. This approach was optimized using standard APTS-labeled oligosaccharides. With the selected (9:1) mixture of 2,5-dihydroxybenzoic acid (DHB) and 5-methoxysalicylic acid (MSA) as matrix and the on-probe sample cleanup procedure with cation-exchange resin, standard APTS-maltotriose was successfully detected down to 50 fmol using linearmode negative MALDI-TOF-MS. Moreover, using extraction delay time, only 100 and 500 fmol of this standard were required, respectively, to obtain accurate reflectron mass measurements and sequence determination through post-source decay experiments. Applied to only 5 µg (294 pmol) of M. tuberculosis ManLAMs, this analytical approach allowed successful mass characterization of the mannooligosaccharide cap structures from the deprotonated molecular ions [M - H]- and the y-type ion fragments obtained in post-source decay experiments. This powerful analytical approach opens new insights into both the characterization of oligosaccharides and the capping motifs displayed by ManLAMs purified from mycobacteria isolated from tubercular patients without in vitro culturing. Lipoarabinomannans (LAMs) are ubiquitously found in the envelope of the Mycobacterium genus1 and contribute to the survival2-7 of virulent mycobacteria in host macrophages.8,9 LAMs are amphipathic glycoconjugates of molecular mass around 17 * Corresponding author. Tel: 33 (0)5 61 17 55 04. Fax: 33 (0)5 61 17 55 05. e-mail: [email protected]. (1) Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64, 29-63. (2) Nigou, J.; Vercellone, A.; Puzo, G. J. Mol. Biol. 2000, 299, 1353-62. 10.1021/ac001368h CCC: $20.00 Published on Web 04/10/2001

© 2001 American Chemical Society

kDa2,10 that consist of a tripartite structure assigned to a mannosylphosphatidyl-myo-inositol anchor, the arabinomannan core, and the capping motifs.8,9 All of the LAMs investigated to date share the same basic structure but are divided into two classes, ManLAMs and PiLAMs, according to the capping motif structures, mannooligosaccharide and phospho-myo-inositol residues, respectively. PiLAMs were identified from a nonvirulent mycobacterial species: Mycobacterium smegmatis,11 whereas ManLAMs have been isolated from Mycobacterium leprae,12 Mycobacterium bovis BCG,10,13 and different strains of Mycobacterium tuberculosis.12 Moreover, the mannooligosaccharide caps are highly implicated in the maintaining of the ManLAMs biological activities.14,15 The structure and the relative abundance of the mannooligosaccharide caps were first determined by mild acid hydrolysis of ManLAMs, fractionation by gel filtration, reducing end tagging by aminobenzoate ethyl ester, and purification by HPLC, followed by fast atom bombardment mass spectrometry (FAB-MS) and FAB-MS/MS analysis.10 Then the complete structures were assigned to mono-, R(1f2)-di-, and R(1f2)-trimannoside units, thanks to NMR and alditol acetate experiments;10,16 however, these (3) Knutson, K. L.; Hmama, Z.; Herrera-Velit, P.; Rochford, R.; Reiner, N. E. J. Biol. Chem. 1998, 273, 645-52. (4) Adams, L. B.; Fukutomi, Y.; Krahenbuhl, J. L. Infect. Immun. 1993, 61, 4173-81. (5) Roach, T. I.; Barton, C. H.; Chatterjee, D.; Blackwell, J. M. J. Immunol. 1993, 150, 1886-96. (6) Chatterjee, D.; Roberts, A. D.; Lowell, K.; Brennan, P. J.; Orme, I. M. Infect. Immun. 1992, 60, 1249-53. (7) Chan, J.; Fan, X. D.; Hunter, S. W.; Brennan, P. J.; Bloom, B. R. Infect. Immun. 1991, 59, 1755-61. (8) Chatterjee, D.; Khoo, K. H. Glycobiology 1998, 8, 113-20. (9) Vercellone, A.; Nigou, J.; Puzo, G. Front. Biosci. 1998, 3, e149-63. (10) Venisse, A.; Berjeaud, J. M.; Chaurand, P.; Gilleron, M.; Puzo, G. J. Biol. Chem. 1993, 268, 12401-11. (11) Gilleron, M.; Himoudi, N.; Adam, O.; Constant, P.; Venisse, A.; Riviere, M.; Puzo, G. J. Biol. Chem. 1997, 272, 117-24. (12) Hunter, S. W.; Gaylord, H.; Brennan, P. J. J. Biol. Chem. 1986, 261, 1234551. (13) Prinzis, S.; Chatterjee, D.; Brennan, P. J. J. Gen. Microbiol. 1993, 139, 264958. (14) Venisse, A.; Fournie, J. J.; Puzo, G. Eur. J. Biochem. 1995, 231, 440-7. (15) Sidobre, S.; Nigou, J.; Puzo, G.; Riviere, M. J. Biol. Chem. 2000, 275, 241522. (16) Chatterjee, D.; Lowell, K.; Rivoire, B.; McNeil, M. R.; Brennan, P. J. J. Biol. Chem. 1992, 267, 6234-9.

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analytical approaches require large amounts of purified ManLAM in the milligram range. More recently, a new analytical approach, based upon the use of capillary electrophoresis (CE) monitored by laser-induced fluorescence (LIF), has been developed.17-19 This process requires (i) ManLAM mild acid hydrolysis, (ii) labeling with 1-aminopyrene1,3,6-trisulfonate (APTS), (iii) direct analysis of the reaction mix with CE-LIF. This strategy allowed the separation and the absolute quantification of the number of different mannooligosaccharide cap units/ManLAM molecule of M. bovis BCG, M. tuberculosis H37Rv/H37Ra, and Erdman mycobacteria strains.2 Their partial structures were then investigated using CE coupled to electrospray triple stage mass spectrometry (ESI-MS). This approach combines the high resolving power of CE for the separation of APTS-oligosaccharide derivatives and the structural information provided by ESI-MS data.20 Indeed, from the mass value of the deprotonated molecular ions obtained, we were able to characterize the mono- and the dimannoside cap motifs that were expected; however, the sensitivity was insufficient for both the ESI-MS characterization of the minor trimannoside cap motif and the MS/MS experiments that could have allowed sequence determination.20 Here we present an alternative approach based upon off-line coupling between CE and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). Indeed, it has been established that APTS tagging increases oligosaccharide ionization.21 MALDI-TOF procedures were first optimized using APTS-tagged standard oligosaccharides to reach high sensitivity by suppressing the multiple cation adducts due to the highly acidic sulfonate groups present on the fluorophore tag that were observed. With the mass spectrometer operating in the linear mode, the detection limits of the deprotonated molecular ions were down to 50 fmol of standard APTS-maltotriose. Moreover, using a 200-ns extraction delay time, only 100 fmol of standard APTSmaltotriose was sufficient to obtain a good signal in the reflectron mode, allowing precise mass measurement of the deprotonated molecular ions. Oligosaccharide sequencing was also achieved using 500 fmol of standard APTS-maltotriose through the y-type fragment ions observed in post-source decay experiments. This approach was successfully applied to the structural characterization of picomolar amounts of the mannooligosaccharide cap motifs from M. tuberculosis H37Rv ManLAMs. EXPERIMENTAL SECTION Bacteria and Growth Conditions. M. tuberculosis H37Rv strain was grown on synthetic Sauton medium as surface pellicles at 37 °C.22 Cells were harvested after 14 days, and the bacterial suspensions were filtered through a 0.22-µm filter before their use for biochemical procedures. (17) Nigou, J.; Gilleron, M.; Cahuzac, B.; Bounery, J. D.; Herold, M.; Thurnher, M.; Puzo, G. J. Biol. Chem. 1997, 272, 23094-103. (18) Gilleron, M.; Nigou, J.; Cahuzac, B.; Puzo, G. J. Mol. Biol. 1999, 285, 214760. (19) Guttman, A.; Chen, F. T.; Evangelista, R. A.; Cooke, N. Anal. Biochem. 1996, 233, 234-42. (20) Monsarrat, B.; Brando, T.; Condouret, P.; Nigou, J.; Puzo, G. Glycobiology 1999, 9, 335-42. (21) Suzuki, H.; Muller, O.; Guttman, A.; Karger, B. L. Anal. Chem. 1997, 69, 4554-9. (22) Romain, F.; Laqueyrerie, A.; Militzer, P.; Pescher, P.; Chavarot, P.; Lagranderie, M.; Auregan, G.; Gheorghiu, M.; Marchal, G. Infect. Immun. 1993, 61, 742-50.

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Reagents and Materials. Mannoheptose and glucose oligomers (maltotriose, maltohexaose) were purchased from Sigma (St. Louis, MO); 1-aminopyrene-3,6,8-trisulfonate (APTS), from Interchim (Montluc¸ on, France). The MALDI matrix, 2,4,6-trihydroxyacetophenone (THAP) was from Fluka (Buchs, Switzerland). 2,5Dihydroxybenzoic acid (DHB) was from Sigma. All of the other matrixes were from Aldrich (Milwaukee, WI). Cation-exchange resin in the NH4+ form was prepared from the H+ form (200400 mesh AG 50W-X8, Bio-Rad, Rockville Center, NY) according to a published procedure.23,24 Cellular ManLAMs from M. tuberculosis H37Rv were purified as described.17 A portion, 5 µg, was then hydrolyzed (0.1 M HCl, 110 °C, for 30 min). The reaction products were then submitted to APTS tagging (see below). APTS Derivatization. The procedure for the reductive amination of oligosaccharides with APTS followed a protocol described elsewhere.25 Dried oligosaccharide standards and hydrolysis products were mixed with 0.4 µL of 0.2 M APTS in 15% acetic acid, and the same volume of a 1 M sodium cyanoborohydride solution was dissolved in tetrahydrofuran. The reaction was performed for 90 min at 55 °C and was quenched by the addition of 20 µL of water. The APTS-derivatized oligosaccharide samples were used directly after derivatization or stored at -20 °C. Capillary Zone Electrophoresis. Capillary zone electrophoresis analysis was performed on a P/ACE 5000 capillary zone electrophoresis system (Beckman Industries, Inc.) with the cathode on the injection side and the anode on the detection side (reverse polarity). Analytical separation was monitored on-column by 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 band-pass filter of 520 nm. UV absorption (254 nm) was used for picopreparative separation. Analytical and picopreparative CE analyses were carried out at 25.0 ( 0.1 °C using 15 mM triethylamine in a 1% (w/v) solution of acetic acid in water, pH 3.5, as the running electrolyte. The applied voltage was set to 20 kV and 10 kV for analytical and micropreparative CE, respectively. The capillary was flushed with 0.1 M HCl. The electropherograms were acquired and stored on a Dell XPS P60 computer using the System Gold software package (Beckman Industries, Inc.). Analytical CE separation was performed using an uncoated fused-silica capillary column (Sigma, Division Supelco, SaintQuentin-Fallavier, France) of 50 µm i.d. and 40 cm effective length (47 cm total length). Samples were loaded by applying a reduced pressure of 0.5 psi (3.45 kPa) for 5 s (6.5 nl injected). For picopreparative CE, an uncoated fused-silica capillary column (Sigma, Division Supelco, Saint-Quentin-Fallavier, France) of 75 µm i.d. and 40 cm effective length (47 cm total length) was used. Samples were loaded by applying a reduced pressure of 0.5 psi (3.45 kPa) for 5 s (14.8 nl injected). Fraction collection was performed using the standard P/ACE 5000 instrument using UV detection. Because the detection window was 7 cm before the exit of the capillary, the precise knowledge of the migration time, obtained when the desired peak had migrated through the detection window, allowed us to precisely calculate the time of (23) Wang, B. H.; Biemann, K. Anal. Chem. 1994, 66, 1918-24. (24) Gibson, B. W.; Engstrom, J. J.; John, C. M.; Hines, W.; Falick, A. M. J. Am. Soc. Mass. Spectrom. 1997, 8, 645-658. (25) Guttman, A.; Pritchett, T. Electrophoresis 1995, 16, 1906-11.

exit of the species that we wanted to collect (estimated by the migration time for the peak, multiplied by the ratio of 7 to 47 cm). The separating voltage was then switched off, and the fractions were eluted by replacing the outer vial with a vial containing 10 µL deionized water and reestablishing the voltage for approximately 1 min, depending on the width of the peak. The CEcollected fractions were then evaporated in a Gyrovap, dissolved again in deionized water and directly used for MALDI-TOF-MS experiments. Sample Preparation and MALDI-TOF Mass Spectrometry. Analysis by MALDI-TOF-MS was carried out on a Voyager DESTR (PerSeptive Biosystems, Framingham, MA) using the linear and reflectron modes. Ionization was effected by irradiation with pulsed UV light (337 nm) from a N2 laser. APTS-derivatives were analyzed by the instrument operating at 20 kV in the negative ion mode using a extraction delay time set at 200 ns. Typically, spectra from 50 to 100 laser shots were summed to obtain the final spectrum. All of the samples were prepared for MALDI analysis using the on-probe sample cleanup procedure with cationexchange resin. The DHB:MSA matrix was prepared using DHB and MSA solutions, each at a concentration of ∼10 mg/mL in acetonitrile/water (7:3 v/v). The solutions were vortexed and sonicated for 5 min. A 90-µL aliquot of DHB solution and 10 µL of MSA solution were combined, then ∼5 mg of NH4+-form cationexchange resin was added, and the mixture was vortexed for 1 min. After letting the beads settle, an aliquot of 60 µL of the supernatant was removed, another ∼5 mg of the NH4+-form cationexchange resin was added, and the mixture was vortexed for 1 min. The matrix solution was freshly prepared daily. Typically, 1 µL of aqueous APTS-derivatized oligosaccharide sample and 1 µL of the matrix solution, containing ∼5 to 10 cation-exchange beads, were deposited on the target, mixed with a micropipet, and dried in a gentle stream of warm air. Post-Source Decay MALDI Mass Spectrometry Experiments. The total accelerating voltage was -20 kV; the reflection voltage was decreased in successive 20% steps, and 10 segments were obtained, each containing the sum of 500 single-shot spectra, and were summed. A 200-ns extraction delay time was used. Mass Calibration. The measurements were externally calibrated at two points with APTS-maltohexaose (m/z 1430.285) and APTS-maltotriose (m/z 944.126). RESULTS AND DISCUSSION Negative Ion Mode MALDI-TOF Analysis of APTS-Sugar Derivatives. For this study, maltotriose APTS tagged by reductive amination25 was selected to optimize the MALDI-TOF experiments. The reaction mix was then directly analyzed by linear MALDI-TOF-MS using the 2,5-dihydroxybenzoic acid (DHB) matrix that is widely used for oligosaccharide MALDI-TOF-MS analysis.26 Without a desalting step, the negative-mode mass spectrum (Figure 1 A) was dominated by APTS-maltotriose deprotonated molecular ions [M - H]- at m/z 944 and APTSmaltotriose molecular sodium and potassium adducts at m/z 966 [M - 2H + Na]-, m/z 982 [M - 2H + K]-, and m/z 988 [M 3H + 2Na]-, in agreement with the literature.21 However, this approach presented at least two major handicaps: (i) the mass (26) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89.

Figure 1. (A) Linear-mode negative MALDI mass spectra of APTSlabeled maltotriose (1 pmol) using the matrix DHB (10 mg/mL) in acetonitrile/water (1:1 v/v) without any desalting step. (B) Linear- and (C) reflectron-mode negative MALDI spectra of APTS-labeled maltotriose with on-probe sample cleanup with cation-exchange beads in the NH4+ form. (B) Linear-mode spectrum (50 fmol); in insert, extension of the peak m/z 944.1, calculated resolution, 1204. (C) Reflectron-mode spectrum (100 fmol) and 200-ns extraction delay time; in insert, extension of the peak m/z 944.1; calculated resolution, 8714. Both spectra were recorded using the comatrix (9:1 v/v) DHB/ MSA in acetonitrile/water (7:3 v/v) with on-probe NH4+ cation exchange. Peaks denoted with asterisk (*) correspond to matrix peaks. The accelerating voltages were -20 kV.

spectrum complexity and (ii) the poor sensitivity in the linear mode, which prevented MALDI-TOF analysis in the reflectron mode. To overcome these problems, we then selected the on-probe sample cleanup procedure using cation-exchange resin in the NH4+ form in order to suppress cation adducts.23,24 This step exchanges alkali metal cations for ammonium ions and favors the formation of the free acid of the sulfonate groups by dissociation.27,28 Indeed, using this desalting step, the negative-ion-mode mass spectrum (data not shown) exhibits only one peak at m/z 944, assigned to the deprotonated molecular ions [M - H]- of the APTS-maltotriose derivative. Moreover, these latter ions were also observed (data not shown) in reflectron-mode analysis using a 200-ns extraction delay time.29 Interestingly, the absence of deprotonated molecular ions [M - H]- for APTS-labeled oligosac(27) Nordhoff, E.; Cramer, R.; Karas, M.; Hillenkamp, F.; Kirpekar, F.; Kristiansen, K.; Roepstorff, P. Nucleic Acids Res. 1993, 21, 3347-57. (28) Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991, 5, 359. (29) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044.

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Figure 2. Analysis of the CE-collected APTS-maltotriose standard. (A) Electropherogram of an aliquot of the collected APTS-maltotriose standard. Capillary, 50 µm i.d. × 47 cm (effective length, 40 cm); buffer, acetic acid 15% (w/v), 15 mM triethylamine in water, pH 3.5, E ) 425 V/cm; detection LIF. (B) Reflectron-mode MALDI mass spectrum of 500 fmol of the CE-collected APTS-maltotriose fraction. The collected fraction was evaporated under vacuum and dissolved in pure water to a concentration of 500 fmol/µL. 1 µL was directly mixed with the matrix on the target probe. The peak at m/z 944.1 corresponds to the deprotonated molecular ion [M - H]- of the APTS-maltotriose, and the peak m/z 864.1 arises from the loss of one SO3 group. Peaks denoted with an asterisk (*) correspond to matrix peaks. See Figure 1C for MALDI-TOF conditions.

charides was reported when the reflectron mode was used without delayed extraction.21,30 Our results confirm that extraction delay time, focusing the broad energy distribution of the analyte ions and minimizing energy loss from collision of ions, is essential to obtain a reflectron signal. However, sensitivity was quite low, because more than 1 pmol of APTS-maltotriose was required to obtain a signal, so other matrixes, such as 2,4,6-trihydroxyacetophenone (THAP),31 6-aza-2-thiothymine (ATT),31 6-hydroxypicolinic acid, coumarine 12032 used alone or mixed with ATT,32 and the mixed matrix 6-hydroxypicolinic acid/3-hydroxypicolinic acid,21 which have been successfully used for MALDI-TOF analysis of acidic glycans, were then investigated for their ability to improve ionization of APTS-labeled oligosaccharides. Nevertheless, in our hands, and as previously reported30 for the MALDI-TOF analysis of ANTS-labeled oligosaccharides, none of these matrixes allowed accurate detection of deprotonated [M - H]- of the APTSmaltotriose standard in the femtomolar range. The best sensitivity was achieved using a 9:1 (w/w) mixture of DHB and 5-methoxysalicylic acid (MSA).33,34 Indeed, Figure 1 shows linear (B) and reflectron (C) mass spectra obtained from 50 fmol and 100 fmol of APTS-maltotriose respectively, using this latter comatrix and on-probe sample cleanup. Both spectra are dominated by the peak at m/z 944.1 assigned to deprotonated molecular ions [M - H]-. In addition to this peak (Figure 1C), a (30) Lemoine, J.; Cabanes-Macheteau, M.; Bardor, M.; Michalski, J. C.; Faye, L.; Lerouge, P. Rapid Commun. Mass Spectrom. 2000, 14, 100-4. (31) Papac, D. I.; Wong, A.; Jones, A. J. Anal. Chem. 1996, 68, 3215-23. (32) Dai, Y.; Whittal, R. M.; Bridges, C. A.; Isogai, Y.; Hindsgaul, O.; Li, L. Carbohydr. Res. 1997, 304, 1-9. (33) Karas, M.; Ehring, H.; Norfhoff, E.; Stahl, B.; Strupat, K.; Hillenkamp, F.; Grehl, M.; Krebs, B. Org. Mass Spectrom. 1993, 28, 1476. (34) Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Anal. Chem. 1995, 67, 1034.

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peak resulting from the loss of one SO3 group was also observed in low amounts at m/z 864.1. This fragmentation was previously noted in the MALDI-TOF analysis of APTS-sugar derivatives21 and heparin-derived oligosaccharides.35 In summary, using the mixed matrix DHB/MSA and on-probe sample cleanup, we were able to analyze down to 50 fmol of APTS-maltotriose derivative using the linear detection mode (Figure 1B), in agreement with the literature.21,30 Moreover, in the reflectron mode and using 100 fmol of APTS-maltotriose, we obtained a peak corresponding to the deprotonated molecular ions [M - H]- with a mass resolution of 8000, leading to a mass accuracy higher than 0.01% (Figure 1C). Similar results were also obtained using a (9:1) mixture of DHB and 1-hydroxyisoquinoline (HIQ),36 but this comatrix was not selected because of its higher chemical background in the lowmass range. APTS-Sugar Derivatives Analysis Using the Combination of CE and MALDI-TOF. The comatrix DHB/MSA and the onprobe sample cleanup procedure with cation exchange beads were then selected to optimize the combination of CE and MALDI-TOFMS with the standard APTS-maltotriose. APTS-maltotriose solution (140 pmol/µL) was loaded at the cathode (14.8 nl, 2.07 pmol injected) and separated according to its electrophoretic mobility. The UV-detected APTS-maltotriose (Figure 2A) was manually collected, and the efficiency of this process was evaluated using CE-LIF by co-injection of an aliquot of the collected solution having a known concentration of standard APTS-mannoheptose. Because only one APTS chromophore per sugar molecule is present, peak integration directly provides a (35) Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-6. (36) Mohr, M. D.; Bornsen, K. O.; Widmer, H. M. Rapid Commun. Mass Spectrom. 1995, 9, 809-14.

Figure 3. MALDI-TOF post-source decay mass spectra of collected APTS-maltooligosaccharide. (A) MALDI post-source decay spectrum of the precursor deprotonated molecular ion [M - H]-, m/z 944.1, from 500 fmol of the CE-collected APTS-maltotriose fraction. (B) MALDI post-source decay spectrum of the precursor deprotonated molecular ion [M - H]-, m/z 1430.3 from 500 fmol of the CE-collected APTS-maltohexaose. See Figure 1C for MALDI-TOF conditions.

molar ratio, thus allowing accurate concentration calculation. The recovery percentage of the collected APTS-maltotriose was almost 90%, underlining the high efficiency of the collection. For the MALDI-TOF-MS analysis of the APTS-maltotriose standard collected by CE, the solution was deposited in a microcentrifuge tube, evaporated to dryness under reduced pressure to eliminate the CE buffer, and dissolved in pure water to the required concentration (500 fmol/µL). Then 1µL of the latter solution was deposited on the MALDI target probe. As already mentioned,21 we found that this process led to a better signal-tonoise ratio in mass experiments then directly analyzing the collected solution. Indeed, Figure 2B represents the MALDI-TOF spectrum obtained in the reflectron mode from 500 fmol of collected APTS-maltotriose using the DHB/MSA comatrix and the on-probe sample cleanup procedure. As expected, the mass spectrum was dominated by an intense peak at m/z 944.1 (Figure 2B) assigned to the deprotonated molecular ions [M - H]allowing post-source decay experiments. Small amounts of the fragment ions at m/z 864.1 resulting from the loss of SO3 groups were also observed. Figure 3A shows the post-source decay-mode spectrum from the deprotonated molecular ions [M - H]- at m/z 944.1 obtained from 500 fmol of the CE-collected APTS-maltotriose. The fragment ions at m/z 620.4 and 782.4 arising from the loss of anhydrodisaccharide and anhydroglucose were assigned to y1 and y2, respectively, according to the nomenclature of Domon (37) Domon, B.; Costello, C. Glycoconj. J. 1988, 5, 397-409.

and Costello.37 The absence of b-type fragment ions was attributed to localization of the negative sulfonate tag charge at the reducing end of the molecule. Following this success, further post-source decay experiments were also successfully conducted on 500 fmol of CE-collected APTS-maltohexaose. As depicted in Figure 3B, the post-source decay spectrum shows five intense peaks corresponding to the y-type fragment ions at m/z 1268.5 (y5), m/z 1106.2 (y4), m/z 944.1 (y3), m/z 782.2 (y2), and m/z 620.4 (y1) arising from the deprotonated molecular ions [M - H]- at m/z 1430.3. These data highlight the performance of the combination of CE and MALDI-TOF-MS for structural analysis of APTS-oligosaccharides. Indeed, reflectron MALDI-TOF-MS allows precise molecular weight measurements, and post-source decay experiments permit complete oligosaccharide sequencing from picopreparative CE. The combination of CE and MALDI-TOF-MS was then applied to the structural characterization of the mannooligosaccharide caps from the ManLAMs of M. tuberculosis H37Rv, which was present in a complex mixture of carbohydrates that were obtained after mild acid hydrolysis of the ManLAM molecules. Structural Analysis by CE/MALDI-TOF-MS of Mannooligosaccharide Caps from the ManLAMs of M. tuberculosis H37Rv. Mannooligosaccharide caps were released by mild acid hydrolysis (0.1 M HCl, 30 min, 110 °C) from 5 µg of cellular ManLAMs from M. tuberculosis H37Rv. Precise ManLAM molarity was monitored by LIF and UV detection as previously described.2 Under these conditions, preferential ManLAM cleavages occurred in the arabinan domain, leading mainly to the formation of arabinose, mannooligosaccharide caps, and mannan core linked to the GPI anchor. These reaction products were then labeled by APTS (see Experimental Section) and analyzed by CE controlled by LIF and UV detection. The LIF electropherogram profile (Figure 4A) shows the presence of six major compounds assigned, in agreement with literature data,17 to free APTS reagent (peak I); Ara-APTS (peak II), which corresponds to the major compound; Man-APTS (peak III); Araf-Ara-APTS (peak IV); Manp-Ara-APTS (peak V); Manp-Manp-Ara-APTS (peak VI); and finally, ManpManp-Manp-Ara-APTS (peak VII). To identify these APTS-sugar derivatives, the reaction mix was directly deposited onto the MALDI plate target without any steps of further separation and using the co-matrix DHB/MSA with the on-probe sample cleanup procedure. Nevertheless, the mass spectrum was dominated by the peak at m/z 590 that corresponds to the deprotonated molecular ions of Ara-APTS (data not shown), which was the major compound present in the mixture, as revealed by CE-LIF analysis (Figure 4A); however, the expected pseudomolecular ions typifying the mannooligosaccharide cap APTS-derivatives were missing. As a result, to eliminate the Ara-APTS compound and to improve the signal of the mannooligosaccharide cap motifs, the three cap motif fractions (peaks V, VI VII, Figure 4A) were collected together in a picopreparative CE step, and 14.8 nl of a hydrolyzed and APTS-derivatized ManLAM solution (120-140 pmol/µL) was loaded on the CE; therefore, the maximum amount of each cap motif per run was 1.9-2.2 pmol of Manp-Ara-APTS, 9-10.5 pmol of Manp-Manp-Ara-APTS and 1.6-1.8 pmol of ManpManp-Manp-Ara-APTS cap motifs. This calculation was based on the relative amounts of each cap motif per ManLAM molecule, evaluated from peak integration (Figure 5A), at 1.1 ((0.1), 5.1 ((0.1), and 0.9 ((0.1), respectively. Because the analytical Analytical Chemistry, Vol. 73, No. 10, May 15, 2001

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Figure 4. (A) Electropherogram of APTS-derivatized oligosaccharides obtained from mild acid hydrolysis (0.1 M HCl, 30 min, 110 °C) of 5 µg of M. tuberculosis ManLAMs and APTS derivatization. Conditions: fused-silica capillary, 50 µm i.d. × 47 cm (40 cm effective length); buffer 1% acetic acid, 15 mM triethylamine, pH 3.6; E ) 425 V/cm; detection LIF. Peak I, free APTS reagent; peak II, corresponding to the major compound, Ara-APTS; peak III, Man-APTS; peak IV, Araf-Ara-APTS; peak V, Manp-Ara-APTS; peak VI, Manp-ManpAra-APTS; and peak VII, Manp-Manp-Manp-Ara-APTS. Peaks denoted with an asterisk (*) correspond to the degradation of APTS reagent. Bracket shows the three mannooligosaccharide caps that were collected together. (B) Electropherogram of the CE-collected fraction containing the three types of mannooligosaccharide caps. (C) Reflectron MALDI-TOF mass spectrum of the collected fraction. See Figure 1C for MALDI-TOF conditions.

sensitivity of the standard APTS-labeled oligosaccharides was in the femtomole range, as expected, a single CE collection yielded enough material for MALDI-TOF experiments. The collected fraction was analyzed by CE-LIF (Figure 4B) and proved to be mainly composed of the three types of mannooligosaccharide caps typified by peaks V, VI, and VII, but traces of Ara-APTS (peak II) were still present. The fraction was then analyzed by negativemode MALDI-TOF-MS using the DHB/MSA comatrix and the on-probe sample cleanup procedure. The reflectron spectrum (Figure 4C) is dominated by the peak at m/z 914.1 that typifies the Manp-Manp-Ara-APTS deprotonated molecular ion [M - H](peak VI in Figure 4B). Likewise, the peaks at m/z 752.1 and 1076.2 were assigned, respectively, to the two other cap motifs Manp-Ara-APTS (peak V in Figure 4 B) and Manp-Manp-ManpAra-APTS (peak VII in Figure 4B). In addition to these compounds, the sensitivity of MALDI-TOF enabled other APTS2328 Analytical Chemistry, Vol. 73, No. 10, May 15, 2001

Figure 5. (A), (B), and (C), electropherograms of the three types of mannooligosaccharide cap that were independently collected. Fractions were reinjected on a fused-silica capillary 50 µm × 47 cm (40 cm effective length); buffer, 1% acetic acid, 15 mM triethylamine in water, pH 3.6, E ) 425 V/cm; detection LIF. In inserts, reflectronmode negative MALDI-TOF mass spectra of each CE-collected APTS-derivatized mannooligosaccharide cap fraction. Each spectrum is dominated by one peak assigned to Manp-Ara-APTS [M - H]-, m/z 752.2 (insert A); Manp-Manp-Ara-APTS [M - H]-, m/z 914.1 (insert B); and Manp-Manp-Manp-Ara-APTS [M - H]-, m/z 1076.2 (insert C). See Figure 1 C for MALDI-TOF conditions.

oligosaccharides corresponding to side products from incomplete acid hydrolysis of the ManLAM molecules also to be unambiguously assigned to the deprotonated molecular ions [M - H]- of Manp-Manp-Araf-Ara-APTS (peak at m/z 1046.2), Manp-Araf-AraAPTS (peak at m/z 884.1), Araf-Araf-Ara-APTS (peak at m/z 854.1), and Araf-Araf-APTS (peak at m/z 722.0). The remaining peaks at m/z 996.1 and 834.1 were also identified as being derived from the molecular ions [M - H]- 1076.2 and 914.1, respectivey, by loss of one SO3 group. In summary, these results demonstrate that the combination of CE and MALDI-TOF-MS is a powerful tool for the characterization of mannooligosaccharide caps in the picomole range in a complex mixture obtained from mild acid hydrolysis of only 5 µg of ManLAMs. It is also important to note that it was not possible to characterize the trimannoside cap motif by CE/ESI-MS from the hydrolysis of 85 µg of BCG ManLAMs,20 so it can be deduced that the combination of CE and MALDI-TOF-MS is, so far, more sensitive than the on-line coupling between CE and ESI-MS for the structural analysis of APTS-derivatized oligosaccharides. Indeed, minor compounds observed in CE-LIF analysis were fully identified by MALDI-TOF-MS. Finally, we collected each mannooligosaccharide cap APTSderivative separately by CE in order to confirm that the peaks observed in MALDI-TOF experiments were representative of a real cap motif molecular entity and not a result of fragmentation. The major problem to overcome was the fact that the collection

of one compound requires turning off the CE separating voltage in order to change the sample collecting microvial at the end of the capillary. During this period, it has already been established that samples which had been previously separated are probably mixed again by diffusion inside the capillary.38 As depicted in Figure 4A, with CE operating at 20 kV, peak IV is separated from peak V by approximately 30 s, so the separating voltage was set to 10 kV, and the mannooligosaccharide caps were separated by >2 min. Under these conditions, the three types of mannooligosaccharide-APTS were independently collected, and the efficiency of the collection was checked by CE-LIF. As seen in Figure 5, the proportion of each cap motif largely predominated in the different electropherograms. Each cap motif collected was then analyzed by MALDI-TOF-MS. As shown in Figure 5 inserts, each reflectron-mode mass spectrum is dominated by one peak typifying the different mannooligosaccharide cap structures. To support these assignments and to demonstrate the pertinence of this approach, we then recorded the post-source decay spectrum of each pseudomolecular ion in order to obtain the y sequencing fragment ions. The different spectra obtained are presented in Figure 6. As noted above for the post-source decay spectrum of APTS-maltotriose and APTS-maltohexaose standards, only y-type ions occurring from the reducing end were observed. Indeed, the PSD spectrum (Figure 6A) of the precursor ions at m/z 914.2, typifying the Manp-Manp-Ara-APTS motif, shows fragment ions at m/z 752.1 (y2) and m/z 590.2 (y1) arising from the loss of anhydro-Man and anhydrodimannoside. Thus, thanks to these y-fragment ions, the sequence Manp-Manp-Ara-APTS was unambiguously confirmed. The y sequencing fragment ions generated from post-source decay experiments on precursor ions at m/z 1076.2 and 752.1 (Figure 6B,C, respectively) were also in complete agreement with the proposed Manp-Manp-Manp-Ara-APTS and Manp-Ara-APTS structures. CONCLUSIONS This report reveals that combining CE and MALDI-TOF/MS generates a powerful tool for the structural analysis of the mannooligosaccharide caps from mycobacterial ManLAMs. From a complex mixture of APTS-tagged carbohydrates obtained after mild acid hydrolysis of 5 µg of M. tuberculosis ManLAM molecules, we were able to collect, on the picopreparative scale, CE-separated APTS-derivatized mannooligosaccharide cap motifs and determine their structures using MALDI-TOF-MS. Elimination of the sodium salt resulting from the reagent used for the reductive amination of sugar with APTS was a key step to obtaining only deprotonated molecular ions, that is, to eliminate sodium molecular ion adducts. The sodium salt was successfully depleted on the MALDI target probe by using cation exchange beads in the ammonium form. Using this sample cleanup procedure combined with the DHB/ MSA co-matrix and extraction delay time, we detected only deprotonated molecular ions with sufficient sensitivity to allow the use of reflectron and post-source decay experiments. Thus, precise molecular weight determination gave the degree of glycosylation, and from the y-type fragment ions observed in postsource decay experiments, the full sequence was established. This work opens the possibility of determining the ManLAMs mannooligosaccharide cap structures of mycobacteria isolated directly (38) Minarik, M.; Foret, F.; Karger, B. L. Electrophoresis 2000, 21, 247-54.

Figure 6. MALDI post-source decay mass spectra of the deprotonated molecular ions present in each CE-collected mannooligosaccharide cap fraction from Figure 6. (A), (B), and (C), [M - H]molecular ions at m/z 914.1, 1076.2, and 752.2 corresponding, respectively, to Manp-Manp-Ara-APTS, Manp-Manp-Manp-Ara-APTS, and Manp-Ara-APTS were selected as parent ions. See Figure 1C for MALDI-TOF conditions.

from infected animals or humans without the need for in vitro culturing and could contribute to the study of the role of these molecules in the adaptation of mycobacteria inside the macrophages. In addition, this analytical approach seems powerful for the structural characterization of the carbohydrate part of glycoproteins. An alternative approach based on on-line coupling Analytical Chemistry, Vol. 73, No. 10, May 15, 2001

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between CE and ESI-nanospray ion-trap MS will also be investigated, because this technology would suppress the collection step. ACKNOWLEDGMENT This work was supported by grants from the Re´gion MidiPyre´ne´es, poˆle agro-alimentaire (RECH/9702343) and poˆle me´dicament (RECH/99001129), from the European Community as part of the Tuberculosis Vaccine Development program and from

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the Ministe`re de l’Education Nationale, de la Recherche et des Technologies (MENRT Microbiologie 9710047). We gratefully acknowledge P. Winterton for checking the English.

Received for review November 21, 2000. Accepted February 13, 2001. AC001368H