Palmitoyl-CoA

Anal. Chem. 1998, 70, 1853-1858

Direct Evidence of Methylglucose Lipopolysaccharides/Palmitoyl-CoA Noncovalent Complexes by Capillary Zone Electrophoresis-Electrospray/Mass Spectrometry Gilles Tuffal,† Anne Tuong,‡ Christiane Dhers,‡ Franc¸ oise Uzabiaga,‡ Michel Rivie`re,† Claudine Picard,‡ and Germain Puzo*,†

Institut de Pharmacologie et Biologie Structurale, CNRS, 205 Route de Narbonne, 31077, Toulouse, France, and Service d’Analyse de Recherche, Sanofi Recherche, 195 Route d’Espagne, 31036 Toulouse, France

Mycobacterial methylglucose lipopolysaccharides (MGLPs) play an important regulatory role in the biosynthesis of long-chain fatty acids by forming complexes with neosynthesized acyl-CoA fatty acid derivatives. The MGLPs from Mycobacterium smegmatis were purified by highperformance anion-exchange chromatography and characterized by LSIMS and CE/ESI-MS. We investigated their interaction with palmitoyl-CoA using capillary zone electrophoresis with both direct and indirect UV detection. In the latter mode, the signal of the UV-transparent MGLPs decreased upon addition of increasing amounts of palmitoyl-CoA; while using direct UV detection, the addition of palmitoyl-CoA to the MGLPs revealed characteristic profiles. The major peak was assigned to the noncovalent MGLP/palmitoyl-CoA complex on the basis of its electrophoretic mobility. The abundance of the complex was found to increase until the MGLP/palmitoylCoA molar ratio reached a 1/1 stoichiometry. The existence of and the stoichiometry of this complex were assessed by CE/ESI mass spectrum analysis, showing pseudomolecular ions of the MGLP/palmitoyl-CoA complex. These results confirm that CE/ESI-MS is a powerful tool to characterize noncovalent molecular association. Mycolic acids are R-branched and β-hydroxylated long-chain fatty acids constituting a hydrophobic cell-wall barrier1 involved in mycobacterial hydrophilic antibiotic resistance and mycobacteria survival in hostile host macrophage cells. Recently, the emergence of Mycobacterium tuberculosis strains that are multidrug-resistant, particularly against isoniazid,2 which target is the mycolic acid biosynthesis,3,4 led to renewed interest in investigating the pathway and the enzymes involved in the mycolic acid * To whom correspondence should be addressed. Tel.: 33 05 61 175504. Fax: 33 05 61 175505. E-mail: [email protected]. † CNRS. ‡ Sanofi Recherche. (1) Jarlier, V.; Nikaido, H. FEMS Microbiol. Lett. 1994, 123, 11-18. (2) Iseman, M. D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2428-2429. (3) Quemard, A.; Lacave, C.; Laneelle, G. Antimicrob. Agents Chemother. 1991, 35, 1035-9. (4) Quemard, A.; Sacchettini, J. C.; Dessen, A.; Vilcheze, C.; Bittman, R.; Jacobs, W. R., Jr.; Blanchard, J. S. Biochemistry 1995, 34, 8235-8241. S0003-2700(97)01101-3 CCC: $15.00 Published on Web 04/01/1998

© 1998 American Chemical Society

biosynthesis. It is now well accepted that the C22 and C24 R-chains are introduced via a Claisen condensation to meromycolic acids.5 These R-chains are synthesized either de novo or by elongation of C16 fatty acids by the fatty acid synthetase I.6 This biosynthesis pathway was found to be stimulated by mycobacterial methylglucose lipopolysaccharides (MGLPs)7 by facilitating the fatty acid release from the fatty acid synthetase I. This property of MGLPs was evidenced by formation of stable complexes between MGLPs and fatty acyl-CoA, allowing the sequestering of the neosynthesized fatty acyl-CoA. The existence of these complexes between MGLPs and C16CoA was previously suggested from gel filtration analysis.8 These data were also supported by proton NMR analysis, showing a shift of MGLP anomeric protons upon addition of fatty acids.9 Finally, the MGLP/C16-CoA dissociation constant (KD) was estimated to be 0.4 µM by fluorometric techniques10,11 and was found to depend on both the MGLPs and the fatty acid structures. In recent years, capillary zone electrophoresis (CE) has been widely used to study noncovalent specific protein-ligand interactions, such as lectin-sugar,12,13 protein-DNA,14,15 proteindrugs,16-18 and antibody-antigen19,20 (for review, see ref 21.) On (5) Goren, M. B.; Brennan, P. J. In Tuberculosis; Youmans, G. P., Ed.; W. B. Saunders Co.: Philadelphia, 1979; pp 63-193. (6) Bloch, K. Adv. Enzymol. 1977, 45, 1-84. (7) Ballou, C. E. Pure Appl. Chem. 1981, 53, 107-112. (8) Machida, Y.; Bloch, K. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 1146-1148. (9) Hindsgaul, O.; Ballou, C. E. Biochemistry 1984, 23, 577-584. (10) Kiho, T.; Ballou, C. E. Biochemistry 1988, 27, 5824-5828. (11) Yabusaki, K. K.; Ballou, C. E. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 691695. (12) Kuhn, R.; Frei, R.; Christen, M. Anal. Biochem. 1994, 218, 131-135. (13) Shimura, K.; Kasai, K. J. Biochem. (Tokyo) 1996, 120, 1146-1152. (14) Stebbins, M. A.; Hoyt, A. M.; Sepaniak, M. J.; Hurlburt, B. K. J. Chromatogr. B, Biomed. Appl. 1996, 683, 77-84. (15) Carter, L. K.; Christopherson, R. I.; dos Remedios, C. G. Electrophoresis 1997, 18, 1054-1058. (16) Hage, D. S.; Tweed, S. A. J. Chromatogr. B, Biomed. Sci. Appl. 1997, 699, 499-525. (17) Busch, M. H.; Carels, L. B.; Boelens, H. F.; Kraak, J. C.; Poppe, H. J. Chromatogr. A 1997, 777, 311-328. (18) Schwarz, M. A.; Neubert, R. H.; Dongowski, G. Pharm. Res. 1996, 13, 11741180. (19) Heegaard, N. H.; Hansen, B. E.; Svejgaard, A.; Fugger, L. H. J. Chromatogr. A 1997, 781, 91-97. (20) Heegaard, N. H. J. Chromatogr. A 1994, 680, 405-412.

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the other hand, the gentle ionization method electrospray ionization/mass spectrometry (ESI) has allowed the detection and the structural characterization of such weakly bound complexes in mass spectrometry.22-24 More recently, combination of those two techniques by direct coupling of CE with ESI-MS has found wide application in biochemical analysis by greatly improving the detection sensitivity and the structural characterization of important biological metabolites present at the trace level (for review, see ref 25). However, to our knowledge, only a few examples are found in the literature which use this technique (CE/ESIMS) for characterization of biological noncovalent complexes.25-27 In the present study, the MGLPs from Mycobacterium smegmatis were purified and characterized by liquid secondary ion mass spectrometry (LSIMS). CE analysis of these purified MGLPs in the presence of palmitoyl-CoA strongly suggested the formation of a noncovalent complex between lipopolysaccharides and the lipidic ligand. This was further confirmed by CE/ESIMS, which clearly demonstrates the presence of these noncovalent complexes between MGLPs and palmitoyl-CoA and allows the determination of the stoichiometry 1:1 for the binding reaction. MATERIALS AND METHODS Extraction and Purification of MGLPs. The extraction from M. smegmatis ATCC 607 and the preliminary purification steps involving both C18 reverse-phase chromatography and Sephadex G-50 gel filtration were performed as previously described for the MGLPs of Mycobacterium xenopi.28 Separation of the MGLPs Mixture by Anion-Exchange Chromatography. Anion-exchange chromatography was performed on a Gilson gradient HPLC system equipped with a Carbopac PA1 column (4 mm × 250 mm), eluted by a linear gradient (0 to 40%) of 500 mM ammonium acetate in deionized water at a flow rate of 1 mL/min. Separation was monitored by pulsed amperometric detection (Dionex Corp.) with 0.5 M sodium hydroxide postcolumn addition at 300 µL/min. Using a split system, only 10% of the sample was directed to the detector while the remaining sample (90%) was collected.29 Analysis of the Methylglucose Polysaccharide (MGP) Cores by High-pH Anion-Exchange Chromatography (HPAEC). The purified MGLP-I, -II, and -III were deacylated with 0.1 M NaOH during 15 min, yielding the MGP cores. The HPAEC analysis was performed on a Dionex DX 300 system equipped with an eluant degassing module, a gradient pump, and a Carbopac PA1 column (4 mm × 250 mm). Eluants were made using (18 MΩ) deionized water (Millipore), 50% dilute NaOH (Baker), and (21) Shimura, K.; Kasai, K. Anal. Biochem. 1997, 251, 1-16. (22) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1991, 113, 8534-8535. (23) Ganem, B.; Li, Y. T.; Henion, J. D. J. Am. Chem. Soc. 1991, 113, 62946296. (24) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. (25) Cai, J.; Henion, J. J. Chromatogr. A 1995, 703, 667-692. (26) Tuong, A.; Uzabiaga, F.; Petitou, M.; Lormeau, J. C.; Picard, C. Carbohydr. Lett. 1994, 1, 55-60. (27) Hamdan, M.; Curcuruto, O.; Di Modugno, E. Rapid Commun. Mass Spectrom. 1995, 9, 883-887. (28) Tuffal, G.; Albigot, R.; Monsarrat, B.; Ponthus, C.; Picard, C.; Rivie`re, M.; Puzo, G. J. Carbohydr. Chem. 1995, 14, 631-642. (29) Delmas, C.; Venisse, A.; Vercellone, A.; Gilleron, M.; Albigot, R.; Brando, T.; Rivie`re, M.; Puzo, G. In Techniques in Glycobiology; Townsend, R. R., Hotchkiss, A. T. J., Eds.; Marcel Dekker: New York, Basel, Hong Kong, 1997; pp 85-109.

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ACS grade sodium acetate (Merck). Eluent A was 0.1 M NaOH, and eluent B was 0.5 M sodium acetate in 0.1 M NaOH. Elution was performed at 1 mL/min and started with 38% of eluent B for 2 min. Then, a concave gradient (7) from 38 to 48% of eluent B was used for 60 min. Detection was monitored using a pulsed electrochemical detector (PED) with a standard wave program for carbohydrate (gold electrode without pH reference, E1 ) 0.1 V (0.5 s), E2 ) 0.6 V (0.1 s), E3 ) -0.6 V (0.05 s)). Purification of the MGPs for LSIMS analysis was performed with the same system but using a semipreparative column (9 mm × 250 mm), eluted at 5 mL/min. Approximately 400 µg of the mixture of MGPs was applied in one run. Desalting of MGLPs and MGPs after Anion-Exchange Chromatography. The MGP fractions were neutralized using HCl (∼1 M) and applied as well as the MGLPs to a C18 T SepPack cartridge (Millipore), which was eluted with water and mixtures of 20% and 70% (v/v) methanol in water. Salts were eluted in the water fraction and in the 20% methanolic water fraction. MGLPs and MGPs were recovered in the 70% methanolic fraction. Finally, complete desalting was achieved by gel filtration chromatography on a P4 biogel (Bio-Rad). LSIMS Analysis of MGLP-I, -II, and -III and Their Respective MGP Cores. MS spectra were recorded on a twosector instrument (ZAB-2E, VG Analytical) in both positive and negative modes. The cesium beam energy was 35 kV, and the accelerating voltage was 8 kV. Full spectra were recorded over a mass range of 800-4000 at a resolution of M/∆M ) 1000. High mass range spectra were recorded over a mass range of 22003200 at a resolution of M/∆M ) 4000. Data were processed on a Dec station 3000/300 (Digital). For each sample, 1 µL of MGP or MGLP at ∼5 µg/µL was mixed with 1 µL of thioglycerol matrix containing 10% of acetic acid on the probe tip. MGLPs Analysis by Capillary Electrophoresis with Indirect UV Detection. All CE-UV analyses were carried out using the PACE 5000 system (Beckman Instruments, Fullerton, CA) equipped with a fused silica column of 47 cm × 50 µm i.d. Anionic separations were typically achieved at +10 kV, using 10 mM benzoic acid, 20 mM sodium phosphate, pH 7, as electrolyte. The detector wavelength was set at 214 nm. Palmitoyl-CoA and MGLP/Palmitoyl-CoA Complex Analysis by Capillary Electrophoresis with Direct UV Detection. Palmitoyl-CoA was purchased from Sigma (purity 92%). Aliquots of palmitoyl-CoA (4 mg/mL in water) were added to a 10-µL sample of MGLP (4 mg/mL) and incubated for 5 min at room temperature before analysis. Final MGLP/palmitoyl-CoA molar ratios were in the range from 0/1 to 2/1. The analyses were conducted at +20 kV using 20 mM sodium phosphate, pH 7, as electrolyte. The detector wavelength was set at 214 nm. MGLP/Palmitoyl-CoA Complex Analysis by CE/ESI-MS. The experiments were conducted on a Platform instrument (Micromass) equipped with an API source. A tricoaxial CE-MS interface (Micromass) was used. A fused capillary of 106 cm × 75 µm i.d. was used for the coupling of the capillary electrophoresis to the mass spectrometer. The sheath liquid was 2-propanol in electrolyte buffer (8/2 v/v). The latter consisted of 20 mM ammonium acetate, adjusted to pH 9 with ammonium hydroxide. Separations were performed at + 30 kV, with an electrospray voltage of -3 kV and

a cone voltage of 55 V. The source temperature was 80 °C. ESI mass spectra in the negative mode were acquired in full-scan mode (200-2700 amu in 10 s). RESULTS AND DISCUSSION Purification and LSIMS Analysis of Native MGLPs. The MGLPs were isolated from M. smegmatis according to Lee30 and Hindsgaul and Ballou9 and fractionated by reverse-phase chromatography according to Tuffal et al.28 The fraction containing the MGLPs was examined for the presence of Glcp, 3-O-Me-Glcp and 6-O-Me-Glcp, routinely identified by GC/MS after methanolysis and derivatization. However, it was found by silicic acid TLC that this fraction was heterogeneous due to the presence of acyl residues assigned to succinyl, acetyl, propionyl, isobutyryl, and octanoyl residues borne in different combinations by the MGLPs.31-33 This fraction was partially resolved by anionexchange chromatography, using a Carbopac PA1 column eluted at neutral pH and monitored by pulsed electrochemical detection. Indeed, it was subdivided in three fractions, MGLP-I, MGLP-II and MGLP-III, according to their retention times (Figure 1A). The MGLP-I, MGLP-II, and MGLP-III were assumed to differ, at least by the number of succinyl residues, tentatively assigned as 0, 1, and 2, respectively, according to the previously described structure.31-33 So, to elucidate their structures, the purified MGLPI, MGLP-II, and MGLP-III were analyzed by LSIMS. Figure 1B represents the MGLP-I positive LSIMS spectrum. The high mass range is characterized by several peaks, separated by 14 mass units, of which the most intense are localized at m/z 3847.5 and 3862.1. By adding KI to the matrix, all these ions were upshifted by 16 Da, allowing their assignment to cationized molecular species (M + Na)+. Deduced from these data, the molecular weights of the two major species are 3824.5 and 3839, respectively. They differ either by the MGP core structure or, as mentioned above, by the acyl appendages.31,32 To determine the contribution of the MGPs to the MGLP-I molecular heterogeneity, the latter was deacylated. The MGP subproducts were purified by HPAEC and analyzed by LSIMS.28,34 The HPAEC chromatogram depicted in the inset of Figure 1B shows the presence of a major MGP. Its molecular weight of 3514 (monoisotopic mass) was determined by LSIMS (data not shown) and is in agreement with 20 Glcps, among which 12 are monomethylated, abbreviated MGP20,12. Besides this MGP20,12, the chromatogram shows two other MGPs in small amounts, assigned, in the same manner, to MGP20,13 and MGP19,12. From these data, it can be advanced that the MGLP-I molecular heterogeneity revealed by the LSIMS spectrum (Figure 1B) arises mainly from a combination of different acyl residues esterifying the MGP20,12 core. MGLP-II was analyzed in the same way. Its LSIMS spectrum (Figure 1C) is dominated by three major peaks at m/z 3932.8, 3947.5, and 3962, assigned again to cationized molecular ions (M + Na)+ which differ by the number of methylenic units. These peaks, compared to the MGLP-I ones, are found upshifted by 100 amu. HPAEC MGP-II analysis shows a chromatographic profile (30) Lee, Y. C. J. Biol. Chem. 1965, 241, 1899-2004. (31) Keller, J.; Ballou, C. E. J. Biol. Chem. 1968, 243, 2905-2910. (32) Dell, A.; Ballou, C. E. Carbohydr. Res. 1983, 120, 95-111. (33) Forsberg, L. S.; Dell, A.; Walton, D. J.; Ballou, C. E. J. Biol. Chem. 1982, 257, 3555-3563. (34) Tuffal, G.; Ponthus, C.; Picard, C.; Riviere, M.; Puzo, G. Eur. J. Biochem. 1995, 233, 377-383.

Figure 1. Separation and LSIMS analysis of the MGLPs from M. smegmatis. (A) Anion-exchange chromatogram using a Carbopac PA1 column monitored by pulsed electrochemical detection. Conditions: gradient of 500 mM ammonium acetate (B) in deionized water, pH 7; a split system permitted only 10% of the sample to be used for detection after postcolumn addition of sodium hydroxide. (B-D) Positive high mass range spectra of MGLP-I, MGLP-II, and MGLPIII, respectively. Conditions: the mass spectrometer was set at a resolution of M/∆M ) 1000 (average masses). Insets: HPAEC analysis of MGP-I (B), MGP-II (C), and MGP-III (D) obtained by alkaline treatment of the respective MGLPs. The first number corresponds to the total number of Glcp units and the second one to the number of mono-O-Me-Glcp units.

(Figure 1C, inset) similar to the MGP-I one which is dominated by the MGP20,12 core structure. This 100 amu mass difference between the MGLP-II and MGLP-I molecular weights agrees with the presence of one succinyl residue in the MGLP-II structure. Finally, the LSIMS mass spectrum of MGLP-III (Figure 1D) appears less complex than the two others, suggesting a lower degree of heterogeneity, as is also confirmed by the HPAEC analysis (Figure 1D, inset). It is dominated by two peaks at m/z 4047.9 and 4062.4, separated by 14 amu, assigned to cationized Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

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Chart 1. Proposed Structure for the MGLP Major Homologues from M. smegmatisa

a MGLP-I, MGLP-II, and MGLP-III contain 0, 1, and 2 succinyl residues (2), respectively. The acyl appendages composition is a combination of acetyl (b), propionyl (b), isobutyryl (b) residues and one octanoyl residue (9). The proposed MGLP structure has been revised at the reducing end (two Glcp(β1f3) branched instead of one Glcp(R1f3) and the other one Glcp(β1f4), and at the linear segment backbone, which is now composed exclusively of (R1f4) linkages, except at the reducing extremity, which is (R1f6).

molecular ions (M + Na)+, which are 100 amu upshifted relative to those in the MGLP-II spectrum, indicating that MGLP-III contains two succinic residues. In summary, Chart 1 represents the proposed structure of the MGLP-I, -II, and -III major homologues, which share the same MGP20,12 polysaccharidic core and differ mainly by the number of succinic residues of 0, 1, and 2, respectively. CE/ESI-MS Analysis of the MGLPs. A mixture of the UVtransparent MGLP-I, -II, and -III was analyzed by CE, monitored by indirect UV detection at 214 nm using benzoic acid as UVabsorbing chromophore in the migration buffer. From preliminary experiments, it was established that, at pH above 4, the MGLPs present an overall net negative charge arising from the ionization of the glyceryl unit localized at the reducing end and of the succinyl residues (Chart 1). Thus, separation can be conducted under anionic conditions using uncoated capillaries in the normal polarity mode (detector at the cathode). Indeed, the electropherogram (Figure 2A) shows three well-resolved peaks at 4.9, 5.4, and 6 min, which were tentatively assigned to MGLP-I, -II, and -III according to their migration order. Since the different MGLPs have close molecular weights (∆M < 10%), the electrophoretic mobility must be higher for MGLP-III containing the largest number of ionizable groups (three): two from the succinic acid residues and one from the glyceric acid unit. MGLP-II contains one of each, while MGLP-I contains only a glyceric acid residue. This attribution was finally assessed by CE/ESI-MS in the negative mode using a 20 mM ammonium acetate run buffer adjusted to pH 9. The TIC electropherogram (m/z 200-2700 amu) shows a profile similar to that obtained with indirect UV detection, characterized by three peaks at 9.4, 10.6, and 12 min, assigned to MGLP-I, -II, and -III, respectively (Figure 2B). The longer analysis time in CE/MS of the MGLPs arises from the difference in capillary length (106 versus 47 cm). The ESI mass spectra of MGLP-I, -II, and -III are dominated by doubly charged pseudomolecular ions (M - 2H)2-. The (MGLP-I - 2H)2- ions are observed at m/z 1918.2 and 1910 (Figure 2C), the (MGLP-II - 2H)2- at m/z 1968.2 and 1962.9 (Figure 2D), and finally the (MGLP-III - 2H)2- at m/z 2017.9 and 2012 (Figure 2E). These data are in agreement with the molecular weights established by 1856 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

Figure 2. Analysis of the MGLP mixture from M. smegmatis by CE and CE/ESI-MS. (A) Indirect UV detection electropherogram; 214 nm. (B) Total ion current (TIC) electropherogram. (C) ESI mass spectrum of MGLP-I. (D) ESI mass spectrum of MGLP-II. (E) ESI mass spectrum of MGLP-III.

LSIMS: for MGLP-I 3839 and 3824.5, for MGLP-II 3939 and 3924.5, and for MGLP-III 4039.4 and 4024.9. In the case of the MGLP-III, the relative abundance of the triply charged pseudomolecular ions (MGLP-III - 3H)3- localized at m/z 1344.8 and 1339.6 is equivalent to that of the doubly charged pseudomolecular ions. Triply charged pseudomolecular ions are also observed in the ESI mass spectra of MGLP-I and MGLP-II but appeared in very low relative abundance. This observation can be explained by the presence of only one and two ionizable groups in MGLP-I and MGLP-II, while this number increases to three in the case of MGLP-III. Study of the MGLP/Palmitoyl-CoA Complexes. To the purified MGLP-I was added different amounts of C16-CoA in molar ratios from 0/1 to 1.2/1. The resulting mixtures were first analyzed by CE using indirect UV detection. Upon addition of palmitoyl-CoA, the intensity of the MGLP-I peak decreased linearly, and the peak finally disappeared when the molar ratio of MGLP/C16-CoA was equal (Figure 3A). This decrease is in good agreement with the formation of a complex between C16-CoA and MGLP-I.

Figure 4. Analysis by CE using direct UV detection of noncovalent complexes of palmitoyl-CoA with MGLP-I, MGLP-II, and MGLP-III in the molar ratio 1/1.

Figure 3. Study by CE of the complex formation between MGLP-I and palmitoyl-CoA (A) using indirect UV and (B-E) using direct UV detection.

To further investigate the existence of these complexes, the CE analysis was also monitored by UV at 214 nm, allowing the detection of the UV-absorbing C16-CoA. The electropherogram of C16-CoA (Figure 3B) shows one peak at 4.9 min assigned to free C16-CoA. As expected from the CoA structure, containing three phosphate groups, the C16-CoA migrates much slower than the MGLP-I. The electropherograms in Figure 3C-E represent the analyses of mixtures of MGLP-I and C16-CoA in the molar ratios 0.5/1, 1/1, and 1.5/1. They show a broad tailing peak having the same migration time (4.9 min) as free C16-CoA, which was thus assigned to unbound C16-CoA. Besides this peak, an additional intense peak was observed at 3.8 min, tentatively assigned to the MGLP-I/C16-CoA complex. Similar results were obtained with MGLP-II and -III. Both electropherograms exhibited a peak of unbound C16-CoA, while the complexed forms of MGLP-II and -III appeared respectively at 4 and 4.2 min (Figure 4). The increase in the migration times from 3.8 to 4.2 min agrees with the number of charges borne by the different MGLPs. These data support the noncovalent association between MGLP-I, -II, and -III with C16-CoA. For a better characterization of these complexes, the analysis was continued using CE/ESI-MS in the negative mode. The Figure 5A represents the reconstructed ion current obtained when equimolar amounts of MGLP-III and C16-CoA were mixed. Mainly three peaks are observed. From mass spectrum analysis, peak 3 at 18.3 min could be ascribed to free C16-CoA, since the ions at m/z 1004.9 and 1026.9 were in accordance with the pseudomolecular ions [C16-CoA - H]- and [C16-CoA + Na - 2H]-, respectively (Figure 6C). The peak 1 at 10.7 min was assigned

Figure 5. Analysis of the MGLP-III/C16-CoA complex by CE/ESIMS. (A) Total ion current from m/z 1000 to 2100. (B) Selective ion monitoring of the pseudomolecular ions (MGLP-III - 2H)2- (m/z 1345.4) and (MGLP-III - 3H)3- (m/z 2019). (C) Selective ion monitoring of the pseudomolecular ion (C16-CoA - H)- (m/z 1004.9). (D) Selective ion monitoring of the complex pseudomolecular ions (MGLP-III/C16-CoA + Na - 3H)2- (m/z 2532.5) and (MGLP-III/C16CoA + Na - 4H)3- (m/z 1689.4).

to free MGLP-III from the ions at m/z 1345.4 and 2019 (Figure 6A). These ions correspond to the multicharged pseudomolecular ions [MGLP-III - 3H]3- and [MGLP-III - 2H]2-, respectively, in agreement with a MGLP-III molecular weight of 4039.4, as established by LSIMS analysis. As expected, this UV-transparent peak was absent in the CE-UV profile (Figure 4, bottom). The ESI mass spectrum of the compound 2 appears more complex (Figure 6B). It is dominated by ions arising both from C16-CoA (m/z 1004.9 and 1026.9) and MGLP-III (m/z 1345.4 and 2019). Analytical Chemistry, Vol. 70, No. 9, May 1, 1998

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Figure 6. ESI high mass range spectra of peak 1, migrating at 10.7 min (free MGLP-III) (A), peak 2 at 15 min (complex) (B), and peak 3 at 18.3 min (free C16-CoA) (C).

But, in contrast to the spectrum of free MGLP-III, two additional small peaks are also observed at m/z 1689.4 and 2532.5. They were assigned to the multicharged pseudomolecular ions of the complex [MGLP-III/C16-CoA + Na - 4H]3- and [MGLP-III/C16CoA + Na - 3H]2-, respectively. These ions are in accordance with the existence of an equimolar complex, MGLP-III/C16-CoA. Within the mass range analyzed (m/z < 2650), no other complex of different stoichiometry was observed. Another interesting observation from Figure 5A, showing the total ion current (TIC) profile, is the presence of a significant ionic current between the first peak at 10.7 min assigned to MGLP-III, and peak 2 at 15 min, attributed to the association between MGLPIII and C16-CoA. This ionic current arises mainly from doubly and triply charged MGLP-III pseudomolecular ions. Indeed, the specific monitoring of these ions (Figure 5B) at m/z 2019 and 1345.4 gives the same profile between 10.6 and 15.2 min as the

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TIC characterized by peaks 1 and 2 and a tailing pattern in between. The ion monitoring of m/z 1004.9 and 1026.9, assigned to [C16-CoA - H]- and [C16-CoA + Na - 2H]-, shows also a tailing pattern but beginning at the migration time of the complex (Figure 5C). These tailing patterns reflect that dissociation of the complex must occur within the capillary, and, once dissociated, each partner will move at its own rate, thus preventing the reassociation that would otherwise normally occur. In addition, dissociation seems also to occur in the ion source, as pseudomolecular ions of the unbound species are observed at the peak migration time of the complex. MGLP-I and MGLP-II were analyzed likewise by CE/ESI-MS. Comparatively, the TIC electropherograms show similar profiles, all characterized by three peaks, assigned to free MGLP, the complex, and free C16-CoA. As previously observed in CE monitored by direct UV, the complexes with MGLP-III, MGLPII, and MGLP-I show decreasing migration times of 15, 13.6, and 12.6 min, respectively. Hence, CE and CE/ESI-MS, which was successfully applied to a study of the association between antithrombine III and anionic pentasaccharide,26 also afford convincing electrophoretic and mass spectrometric evidence, confirming the existence of noncovalent complexes of MGLPs with palmitoyl-CoA in the stoichiometry of 1/1. In the future, this analytical approach can be developed in order to determine affinity constants, in the micromolar range, of structurally different MGLPs with long-chain fatty acids. This knowledge will allow us to precisely determine the topologic mechanisms involved in the recognition of MGLPs by long-chain fatty acids which regulate their biosynthesis by the fatty acid synthetase I. ACKNOWLEDGMENT We thank R. Albigot for technical assistance. This work was supported by grants from Ministe`re de l’ Education Nationale, de l’ Enseignement Superieur, de la Recherche, et de l’Insertion Professionnelle ACC SV6/9506005 and from the region MidiPyre´ne´es, RECH/9407528. Received for review October 3, 1997. Accepted February 4, 1998. AC971101R