Glycobiology Advance Access originally published online on January 22, 2007
Glycobiology 2007 17(4):367-373; doi:10.1093/glycob/cwm006
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Purification and structural characterization of de-N-acetylated form of GD3 ganglioside present in human melanoma tumors
2 Laboratory of Dermatological Research, University of Lyon-1 and Edouard Herriot Hospital, 69437 Lyon Cx 03, France
3 Laboratory of Biological Chemistry, CNRS UMR 8576, USTL, 59655 Villeneuve d'Ascq Cx, France
4 Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan
5 Department of Dermatology, Hotel-Dieu Hospital, Lyon, France
6 Institute of Macromolecular Chemistry Petru Poni, Aleea Gr. Ghica Voda 41A, Iassy 6600, Romania
1 To whom correspondence should be addressed; Tel: +33-4 72 11 03 07; Fax: +33-4 72 11 02 90; e-mail portoukalian{at}lyon.inserm.fr
Received on May 6, 2006; revised on December 22, 2006; accepted on January 11, 2007
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Abstract |
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The presence of gangliosides containing de-N-acetylated sialic acids in human tissues has been so far shown by using mouse monoclonal antibodies specific for the de-N-acetylated forms, but the isolation and chemical characterization of such compounds have not yet been performed. Since indirect evidence suggested that de-N-acetylGD3 ganglioside could be present in human melanoma tumors, we analyzed the gangliosides purified from a 500-g pool of those tumors. The de-N-acetylGD3 that was found to migrate just below GD2 in thin-layer chromatography was isolated from the disialogangliosides by high-pressure liquid chromatography using the specific antibody SGR37 to monitor the elution. The amount of antigen was found to be 320 ng per gram of fresh tumor or 0.1% of total gangliosides. Gas chromatography–mass spectrometry analysis of the antibody-positive ganglioside showed that sialic acids were formed of one molecule of N-acetylneuraminic acid and one molecule of neuraminic acid. Radioactive re-N-acetylation of the antigen yielded a GD3-like ganglioside with the radioactive label on the external sialic acid. The constitutive fatty acids were found to differ markedly from those of GD3 and 9-O-acetylGD3 isolated from the same pool of tumors. The major fatty acids were C16:0 and C18:0 in de-N-acetylGD3, whereas GD3 and its 9-O-acetylated derivative contained a large amount of C24:1. These data show that de-N-acetylGD3 ganglioside is indeed present in human melanoma tumors, and the fatty acid content suggests the existence of a de-N-acetylase mostly active on the molecular species of gangliosides with short-chain fatty acids.
Key words: melanoma / gangliosides / GD3 / de-N-acetylation / mass spectrometry
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Introduction |
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Gangliosides are amphiphilic components of the plasma membranes made of a sialylated oligosaccharide coupled to a ceramide. They are located at the external surface of the outer leaflet and involved in many biological processes (Nagai and Iwamori 1995
). Malignant transformation induces major changes in the biosynthetic pathways of glycosphingolipids in human tissues (Hakomori and Kannagi 1983), leading to the appearance of tumor-associated gangliosides that have been used as serum markers (Portoukalian et al. 1978) and as target antigens for immunotherapy (Portoukalian et al. 1991). The role of gangliosides is mostly dependent on the carbohydrate composition of their oligosaccharide moiety. Slight alterations in the structure of the constitutive sialic acids, such as hydroxylation or O-acetylation, can result in striking changes in their biological properties (Schauer 1991).
Whereas O-acetylation of sialic acids bound to gangliosides has been extensively studied because of the appearance of O-acetylated compounds in tumor tissues, the mechanisms leading to de-N-acetylation of sialic acids have been much less investigated (Hanai et al. 1988; Manzi et al. 1990; Sjoberg et al. 1995; Chammas et al. 1999). The finding that both O-acetyl and N-acetyl groups of ganglioside-bound sialic acids turnover faster than the parent molecules strongly supports the existence of de-N-acetylated ganglioside species (Manzi et al. 1990). Although these studies suggested the presence of de-N-acetylated ganglioside-bound sialic acids in addition to the identification of a de-N-acetylated from of GM1 ganglioside in bovine brain (Hidari et al. 1993), such compounds in human tissues remain to be chemically characterized. Using a method involving the formation of heptafluorobutyrate derivatives of the methyl esters of sialic acids, a minor constituent in ovine submaxillary mucin yielded a mass spectrum characteristic of neuraminic acid (Zanetta et al. 2001). Since this sialic acid was suggested to be present in gangliosides from human melanoma (Chammas et al. 1999), we analyzed the gangliosides purified from a large pool of human melanoma tumors and revealed by specific antibodies. The present study details the structure of a GD3 ganglioside in which sialic acids are made of one molecule N-acetylneuraminic acid (Neu5Ac) and one de-N-acetylneuraminic acid (Neu5NH2).
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Figure 1 shows the isolation of individual gangliosides from the disialoganglioside fraction of human melanoma tumors using high-pressure liquid chromatography (HPLC). As shown in Figure 2, the selective elution of tumor-associated de-N-acetylGD3 was monitored by immunostaining the collected fractions with the specific monoclonal antibody (mAb), SGR37 (Sjoberg et al. 1995
), using synthetic de-N-acetylGD3 as a standard. The final recovery of pure de-N-acetylGD3 was 160 µg from the 500 g pool of fresh melanoma tumors that yielded 145 mg of total gangliosides, as figured out from the amount of assayed sialic acid.
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The putative de-N-acetylGD3 did not react with anti-GD3 mAb 4F6, but upon re-N-acetylation, the ganglioside migrated like GD3 and was stained by the Mab 4F6 (not shown). Following re-N-acetylation of de-N-acetylGD3 purified from melanoma with radioactive acetic anhydride and cleavage of the labeled GD3 using Vibrio cholerae neuraminidase for 30 min, the resulting GM3 visualized by immunostaining with a specific antiserum was not radioactive (Figure 3). This result suggests an external position of the neuraminic acid in the de-N-acetylGD3 molecule.
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As shown in Figure 4A, gas chromatography-mass spectrometry (GC-MS) analysis indicated that the de-N-acetylGD3 ganglioside isolated from human melanoma contained only two peaks corresponding to sialic acids. The first corresponded to Neu5Ac and the second to Neu5NH2. No traces of Neu5Gc and mono- and poly-O-acylated sialic acids were present. The quantity of Neu5Ac was apparently higher than that of Neu5NH2 on the total ion current chromatogram, but this was due to a contamination by a compound not containing helpta fluoro butyrate (HFB) groups. In fact, the quantities of the two compounds revealed, Neu5Ac and Neu5NH2, using the ion at m/z = 169 characteristic of HFB groups were identical within a 1% error. The two compounds were characterized by their mass spectra. The nature of the derivative of Neu5Ac was characterized by its typical mass spectrum (Figure 4B) The nature of the Neu5NH2 derivative was ascertained by the presence of an intense ion at m/z = 505, indicating the presence of one O-HFB and one N-HFB groups on the pyranic ring (Figure 4C). On the basis of literature data, Neu5NH2 was never detected using the classical sensitive techniques of analysis for sialic acids. One possible explanation was that the free amino group of C(5
) formed lactam with C(1) carboxyl group (Mitsuoka et al. 1999), which is quite stable and would not be displaced during the acylation by HFB anhydride (HFBAA) to yield the intense ion seen at m/z = 505. The absence of detection of Neu5NH2 was more likely due to the formation of a cyclic imine between the free amino group of the C(5) carbon atom and the keto group of the C(2) carbon atom. This relatively stable imine (theoretically in equilibrium with the amino form) could not be changed to amino form in anhydrous conditions used for the formation of trimethlylsilyl (TMS) derivatives; the TMS derivatives of amino groups are also extremely unstable. The alternative could be the pyridine-catalyzed acetylation of the free amino group, which was commonly used to re-acetylate free amino groups of neuraminic acid (Neu) liberated from glycoconjugates by acid-catalyzed methanolysis. But, it should be stressed that when Neu was liberated by methanolysis, it was not liberated as neuraminic methyl ester but as the O-methyl glycoside of the methyl ester of Neu5NH2, this compound being unable to produce the cyclic imine described above. Furthermore, Pyridine was not sufficiently electronegative to ensure a rapid change of the cyclic imine into the cyclic amino form. The reason for the detection of Neu5NH2 by GC-MS of HFB derivatives was that HFBAA attacked preferentially free amino groups than hydroxyl groups. This was actually observed for amino acids. The overall composition of this ganglioside (monosaccharide, fatty acids and long-chain bases) was verified using GC-MS of the same sample, previously submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. The molar ratio of the constitutive monosaccharides Glc, Gal, Neu5NH2, and Neu5Ac was 1:1:1:1. These data were not contrasting with the previous ones since acid-catalyzed methanolysis provoked de-N-acetylation of Neu5Ac together with the formation of the O-methyl glycoside, unable to form the cyclic imine. Consequently, these data indicated that the compound was a de-N-acetylGD3 ganglioside. The long-chain base composition of de-N-acetylGD3 showed that d18:1 sphingenine (sphingosine) was the major sphingoid base, along with trace amount of d18:0 sphinganine, as in GD3 (not shown). The fatty acid composition of de-N-acetylGD3 was also analyzed by gas–liquid chromatography (GLC) and compared to those of GD3 and 9-O-acetylGD3 gangliosides purified from the same pool of melanoma tumors (Figure 5). As shown in Table I, de-N-acetylGD3 was characterized by a high content of C16 and C18 fatty acids, whereas both GD3 and its 9-O-acetyl derivative contained mostly long-chain fatty acids, in agreement with the relative amounts of the ganglioside doublets visualized by thin-layer chromatography (TLC) (Figures 1 and 2).
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Discussion |
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A De-N-acetylated ganglioside was first reported by Hanai et al. (1988)
by the isolation of de-N-acetyl-GM3 from the monosialoganglioside fraction of A431 cells and B16 melanoma cells. This type of membrane ganglioside strongly enhanced the kinase activity associated with the receptor of epidermal growth factor. Re-acetylation experiments suggested that such a compound was present in the membranes of melanoma cells (Manzi et al. 1990). The presence of de-N-acetyl-GM3 and de-N-acetyl-GD3 was further evidenced by the production of mAbs against synthetic compounds (Sjoberg et al. 1995). De-N-acetylGD3 had a diffuse intracellular location and was efficiently internalized into a compartment that is distinct from lysosomes. Rounding up of melanoma cells occurring during growth in culture was associated with the relocation of the intimal pool of de-N-acetyl-GD3 to the cell surface. Although these experimental data favored the existence of Neu5NH2 in melanoma cells, there was not yet direct evidence for the presence of this molecule in human gangliosides. Using GC-ion mobility spectrometry (GC–IMS) analysis of HFB derivatives (Zanetta et al. 1999), Neu5NH2 was recently identified at a significant level (5% of Neu5Ac) in ovine submaxillary mucin (Zanetta et al. 2001). In the electron impact (EI) mode of ionization, this compound is characterized by the presence of an intense ion at m/ez = 505, corresponding to the pyranic ring substituted with two HFB groups, respectively, on the alcohol group and on the amino group of the C(4) and C(5) carbons. This intense ion was almost specific for this compound, in such a way that quantities of 1 pg injected onto the column of the GC–IMS apparatus could be unambiguously detected in a complex mixture containing several nanograms of other sialic acids. The presence of this compound did not correspond to an artifact since it was absent from the mild hydrolysis products of Neu5Ac or Neu5Gc and was completely absent from gangliosides and glycoproteins of rat brain synaptosomal plasma membranes (Zanetta JP, unpublished). The ganglioside analyzed here presented a very peculiar composition since the ratio of the recorded peaks of Neu5Ac and Neu5NH2 was 1:0998. This indicated that this compound contained one Neu5NH2 and one Neu5Ac per molecule. The carbohydrates were Glc, Gal, Neu5NH2 and Neu5Ac, with a molar ratio of 1:1:1:1, leading to the identification of gaglioside as de-N-acetylGD3. Although the long-chain base composition was similar to that of GD3 ganglioside of human melanoma tumors, the fatty acid pattern was strikingly different, with C16:0 and C18:0 as major species in the de-N-acetylGD3 molecule, whereas C24:1 as major fatty acid in GD3 and 9-O-acetylGD3 molecules of human melanoma tumors. It has been suggested that 9-O-acetylGD3 and de-N-acetylGD3 derived from GD3 through the activity of respectively specific O-acetyltransferase and de-N-acetylase (Manzi et al. 1990). Our data suggest that the latter enzyme has a specificity restricted to molecular species containing C16 and C18 fatty acids, whereas the O-acetyltransferase uses all molecular species of GD3 as substrates. It would be interesting to study the possibility that other major gangliosides of human tumors are substrates for the de-N-acetylase. For example, GD2 that has been found as an O-acetylated derivative in human melanoma (Sjoberg et al. 1992) might be also present in its de-N-acetylated form. In the light of the study by Ecsedy et al. (1998) showing that tumor-infiltrating macrophages influence the glycosphingolipid composition of mouse tumors, the possibility should be considered that de-N-acetylGD3 actually derived from the tumor-infiltrating lymphocytes. This could account for the differences in fatty acid composition between GD3 and de-N-acetylGD3 in the melanoma tumors analyzed in our study, since it is known that C16:0 is a major fatty acid of glycosphingolipids of human lymphocytes (Lee et al. 1981). In any case, the potential role of de-N-acetylated gangliosides as tumor markers emphasized by Chammas et al. (1999) and the biological properties reported for de-N-acetylGD3 in melanoma cells (Sjoeberg et al. 1995) should boost additional investigations in this field. It is likely that many glycosphingolipids whose structures have not yet been characterized are present in minute amounts in tumors. These compounds might play important roles in the biology of malignant cells. Provided that large amounts of tumors can be made available, the ongoing development of more sensitive methods of structural analysis should lead in a near future to the isolation and characterization of novel cancer-associated sphingolipids.
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Material and methods |
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Tissues
Human melanoma tumors were surgically removed at the Department of Dermatology, Hotel-Dieu Hospital, Lyon, France (Pr. Luc Thomas), and proved to be malignant melanoma by histopathological examination. The excised tumors were snap frozen in liquid nitrogen and stored at –80 °C until use.
Purification and fractionation of gangliosides
A 500-g pool of melanoma tumors were cut into small pieces, then homogenized in chloroform–methanol mixture (1:1, v/v) using Polytron homogenizer (Kinematica, Lucern, Switzerland). The tissues were extracted with 5 L of the same solvent by stirring overnight at room temperature. After filtering, the solvent-treated tissue was extracted again by stirring 3 h at room temperature with 2.5 L of chloroform–methanol mixture (1:2 v/v), then filtered again. The solvent extracts were pooled and evaporated to dryness at 40 °C under vacuum with a rotary evaporator. The lipid residue was taken up with 250 mL of 1:1 (v/v) chloroform–methanol mixture and filtered to remove the nonsoluble particles. The lipids were partitioned after addition to the filtrate of phosphate-buffered saline (PBS), pH 7.4 to obtain a ratio of chloroform–methanol–PBS at 1:1:0.7 (by volume) (Bouchon et al. 1990). Following three successive partitions, the pooled upper phases were loaded onto styrene–divinylbenzene copolymer columns (ENVI-CHROM, Supelco, L'Isle d'Abeau, France) and the gangliosides were recovered as described (Popa et al. 2002). The lipid-bound sialic acid was assayed by a microscale method adapted from the periodate–resorcinol method (Jourdian et al. 1971). The ganglioside profile was determined by high-performance TLC (HPTLC) on aluminium-backed silica gel 60 plates (Merck, Darmstadt, Germany) developed in chloroform–methanol–0.22% aqueous calcium chloride (55:45:10) (by volume) (solvent I). Visualization was done with the resorcinol–HCl spray reagent (Svennerholm 1963). The melanoma gangliosides were separated according to their number of sialic acid residues by ion-exchange column chromatography on DEAE-Sephadex A-25 (Pharmacia, Uppsala, Sweden) using increasing concentrations of ammonium acetate in methanol according to Yu and Ledeen (1972). The monosialo-, disialo- and trisialoganglioside fractions were evaporated to near dryness, taken up in the volume of distilled water necessary to obtain a 0.1 M solution of ammonium acetate, then the fractions were desalted on copolymer columns (Popa et al. 2002). Individual gangliosides were isolated from the disialoganglioside fraction by HPLC on a 250-10 Si100 column (Macherey-Nagel, Germany) with a L-6200 apparatus (Hitachi, Tokyo, Japan) using a ternary gradient of hexane–isopropanol–0.05% aqueous potassium acetate (55:36:9 to 55:30:15, by volume) at a flow rate of 2 mL/min. Potassium acetate was necessary since we previously reported that the presence of salts prevents the strong interaction of de-N-acetylated gangliosides with the silica gel of the HPLC column, thereby improving the recovery of such compounds (Rebbaa and Portoukalian 1995). Fractions of 2 mL were collected and elution was monitored by TLC in solvent I after spotting 50 µL of each fraction on HPTLC plates under a stream of nitrogen with a Linomat IV apparatus (Camag, Mettenz, Switzerland). The melanoma-associated gangliosides were identified by immunostaining on TLC plates (ITLC), using the murine IgG3 mAbs, 7H2 (Dumontet et al. 1997), and 4F6 (Thomas et al. 1995) and 4G2 (Portoukalian et al. 1993), respectively, specific for 9-O-acetylGD3, GD3 and GD2. Fractions containing pure 9-O-acetylGD3 and GD3 were pooled in a flask and evaporated using a rotary evaporator. The elution of de-N-acetylGD3 from the HPLC column was monitored by ITLC using the specific mAb SGR37 (Sjoberg et al. 1995). The SGR37-reactive fractions were pooled.
Synthesis of de-N-acetylGD3
Standard de-N-acetylGD3 was prepared by alkaline hydrolysis of authentic GD3 with 0.1 N KOH in 90% n-butanol at 80 °C for 4 h (Nores et al. 1988). After neutralization with ethyl acetate and evaporation under nitrogen, the resulting mixture was taken up in 1 mL of 2:1 (v/v) ratio of chloroform–methanol mixture, filtered and loaded on a 250-4 Si-100 column (Merck, Darmstadt, Germany) to isolate de-N-acetylGD3 by HPLC on a Hitachi-L-6200 apparatus using an isocratic gradient of isopropanol–hexane–water 55:34:11 at a flow rate of 0.25 mL/min, with fractions collected every 2 min. The solution of hydrolyzed gangliosides neutralized by ethyl acetate was not desalted before HPLC, for the same reason as detailed in the Purification and fractionation of gangliosrdes section. Elution of the chemically produced de-N-acetylGD3 was monitored by ITLC using the specific mAb SGR37. The antibody-positive HPLC fractions were pooled, evaporated to dryness and assayed by the periodate–resorcinol method (Jourdian et al. 1971).
Immunochemical characterization of the melanoma-associated de-N-acetylGD3
The SGR37-reactive fractions collected after HPLC of the disialogangliosides of human melanoma tumors were pooled, evaporated to dryness and spotted on an aluminium-backed HPTLC plate, along with standards of GD2 and of chemically produced de-N-acetylGD3 that were spotted on separate lanes. The plate was migrated twice in solvent I, then the lanes containing the standards were cut out and visualized with resorcinol–HCl. The remaining plate was sprayed with primulin 0.01% in acetone and visualized under ultraviolet light. The silica gel area corresponding to the migration of standard de-N-acetylGD3 was scrapped into a test tube, with care to avoid contamination of the sample with GD2 ganglioside. The tube was filled with 1:1 (v/v) ratio of chloroform–methanol mixture and sonicated for 2 min in a bath sonicator, then left for 30 min at room temperature to elute the gangliosides adsorbed onto the gel. After centrifugation at 3000 rpm to pellet the silica gel particles, the supernatant was transferred to an other tube and evaporated to dryness under nitrogen. The residue was taken up in methanol and, after addition of an equal volume of PBS, PH 7.2, the ganglioside was purefied from nonlipidic contaminants by reverse-phase chromatography on a styrene–divinylbenzene copolymer column. The purity of the SGR37-reactive ganglioside was assessed by ITLC using mAb SGR37 specific for de-N-acetylGD3 that was found to be positive. Neither anti-GD3 mAb 4F6 to GD3 nor anti-GD2 mAb 4G2 showed any reactivity. The purified SGR37-reactive ganglioside was assayed by a microscale adaptation of the periodate–resorcinol method. Following re-N-acetylation of an aliquot with acetic anhydride as described (Rebbaa and Portoukalian 1995), the reactivity of the resulting re-N-acetylated compound was tested with mAbs SGR37 and 4F6, respectively, specific for de-N-acetylGD3 and GD3.
Determination of the position of the neuraminic acid in the de-N-acetylGD3 molecule
In order to determine the position of the neuraminic acid in the molecule of de-N-acetylGD3, an aliquot was re-N-acetylated with tritiated acetic anhydride (ARC, Fleurus, Belgium). Then, 10 µg of standard unlabeled GD3 were added to the tube containing the radioactive compound and after evaporation of the solvent, the gangliosides were taken up in 200 µL of 0.05 M acetate buffer, pH 5.2, containing 0.1 U of Vibrio cholerae neuraminidase (Sigma, L'Isle d'Abeau, France). After 30 min at 37 °C, 200 µL of methanol were added and the ganglioside solution was loaded on a copolymer column. The column was washed with 5 mL of distilled water, then eluted with methanol and chloroform–methanol. After evaporation to dryness of the eluates, the gangliosides were taken up in chloroform-methanol 2:1 (v/v) and migrated on an HPTLC plate in solvent I, along with standard gangliosides GM3 and GD3 radioactively labeled by de-N-acetylation and re-N-acetylation with tritiated acetic anhydride. The radioactive spots were detected by autoradiography, then the gangliosides were visualized with resorcinol–HCl reagent. The structure of de-N-acetylGD3 purified from melanoma tumors was then analyzed by GC-MS as described in the GC-MS analysis section.
Cleavage of sialic acid residues from gangliosides and preparation of the samples for derivatization
All experiments were performed in heavy-walled Pyrex tubes with a Teflon-fined screw cap. Sialic acids were liberated from glycolipids using 2 M acetic acid for 105 min at 80 °C. The tubes containing glycolipid samples were evaporated under vacuum at room temperature then supplemented with 2 M acetic acid (at least 200 µL/10 µg glycolipid). The tightly closed vials were gently shaken and placed in an oven at 80 °C. After 105 min, the samples were cooled and evaporated to dryness using a rotary evaporator (with a Teflon-lined fitting to the reaction vessels) at room temperature.
Derivatization of sialic acids
The dry samples (0.1–1 µg of total sialic acid) were taken up with 100–200 µL of anhydrous methanol at the bottom of the vial (in order to partially dissolve sialic acids), followed by the addition of 200 µL of a diazomethane solution in diethyl ether, then the tubes were tightly closed. And the samples were left for 4 h at room temperature without stirring. The reagents were evaporated to dryness under a stream of nitrogen in a ventilated hood, and then supplemented with 200 µL acetonitrile and 25 µL HFBAA (Zanetta et al. 2001). The closed vials were heated for 5 min at 150 °C in a sand bath, cooled at room temperature and the samples were evaporated in a stream of nitrogen in a ventilated hood. The residue was solubilized into 200 µL of acetonitrile previously dried on calcinated calcium chloride and 1 µL aliquots were injected onto the Ross injector of the GC–MS apparatus.
Simultaneous determination of the monosaccharide, fatty acid, and long-chain base composition of the ganglioside
After GC–MS analysis of sialic acids, the samples were evaporated to dryness under a light stream of nitrogen and supplemented with 250–500 µL of the methanolysis reagent (0.5 M HCl in anhydrous methanol) and the closed vessels were left for 20 h at 80 °C.
After methanolysis, samples were evaporated to dryness under a light stream of nitrogen in a ventilated hood, then 200 µL of acetonitrile and 25 µL of HFBAA were added. The closed vessels were heated for 15 min in a sand bath at 150 °C. When an analysis is to be performed, the samples are heated again for 5 min at 150 °C, cooled, evaporated in a light stream of nitrogen in a ventilated hood, then taken up in the appropriate volume of acetonitrile. An aliquot of the acetonitrile solution of the HFB derivatives (containing <2 ng of each compound) was introduced in the Ross injector of the GC–MS apparatus (Zanetta et al. 1999).
GE–Ms analysis
For GC–MS analysis, the GC separation was performed on a Carlo Erba GC 8000 gas chromatograph equipped with a 25 m x 0.25 mm CP-Sil5 CB Low-bleed MIS capillary column, 0.25 pm film phase (Chrompack France, Les Ulis, France). The temperature of the Ross injector was set at 260 °C and the samples were analyzed using temperature programming at 90 °C for 3 min then 5 °C/min until 260 °C, followed by a plateau of 20 min at 260 °C. The column was coupled to a Finnigan Automass Il mass spectrometer (mass limit 1000) for routine analyses. In order to verify masses higher than 1000, the coupling was to a Riber 10-10 OH mass spectrometer (mass detection limit 2000). Analyses were performed routinely in the El mode (ionization energy 70 eV; source temperature 150 °C).
Analysis of fatty acids by GLC
The fatty acid compositions of de-N-acetylGD3, 9-O-acetylGD3, and GD3 were determined by GLC after acid hydrolysis of purified gangliosides carried out for 20 h at 80 °C in 0.8 N HCl in anhydrous methanol. Fatty acids were extracted with hexane and C21:0 methyl ester was added as an internal standard. The fatty acids were analyzed on a HP-1 (Hewlett-Packard) capillary column (25 m x 0.32 mm) with temperature increasing at 3 °C min from 175 °C to 310 °C on a HP-5890 series II apparatus (Hewlett-Packard, Lyon, France) equipped with a flame ionization detector and coupled to a CR3A data recorder (Shimadzu, Kyoto, Japan). The fatty acids were identified by comparison of their retention times with those of authentic standards (Supelco, L'Isle d'Abeau, France).
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Conflict of interest statement |
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None declared.
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Abbreviations |
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EI, electron impact; GC–IMS, gas chromatography–ion mobility spectroscopy; GC–MS, gas chromatography-mass spectrometry; GLC, gas–liquid chromatography; HFB, heptafluorobutyrate; HPLC, high-pressure liquid chromatography; HPTLC, high-performance thin-layer chromatography; ITLC, immunostained thin-layer chromatography; mAb, monoclonal antibody; Neu, neuraminic acid; Neu5Ac, N-acetylneuraminic acid; Neu5NH2, de-N-acetyl-neuraminic acid; PBS, phosphate-buffered saline; TLC, thin-layer chromatography; TMS, trimethylsilyl.
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References |
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