2Laboratoire de Chimie Biologique, CNRS UMR 8576, 59655 Villeneuve dAscq Cedex France, and 3Biochemisches Institut, Universität Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany
Received on January 16, 2001; revised on March 20, 2001; accepted on March 21, 2001.
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Abstract |
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Key words: Kdn/lactone/lactyl/methyl/sulfate
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Introduction |
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Several methods were developed for the identification and quantitative determination of these compounds. The use of fluorescent labeling specific for 2-keto carboxylic acid (Hara et al., 1989; Klein et al., 1997
; Iwersen et al., 1998
) followed by the separation of the compounds by high-performance liquid chromatography (HPLC) provided a very sensitive method. However, the separation power of HPLC was still insufficient for resolving very complex mixtures, whereas coupling with mass spectrometry (MS) was difficult for routine analyses. Furthermore, this method could not reveal possible lactones of sialic acids, which might play important biological roles. In contrast, gas chromatography (GC)/MS methods using the derivatization of the methyl esters of sialic acids as trimethyl-silyl (TMS) ethers, allowed easy GC/MS analyses (Kamerling et al., 1975
; Schauer et al., 1976
; Kamerling and Vliegenthart, 1982
). However, these methods also suffered from some difficulties. One was that, when applied to biological samples presenting different impurities, the silylation reaction had a poor yield and the derivatives were unstable. The other was that the TMS derivative of the semi-acetalic group was unstable and was partially lost by pyrolysis in the injector of the gas chromatograph in a poorly reproducible way. Consequently, instead of the two peaks of the anomers of each sialic acid, four peaks were produced, as already observed for glucosamine (Maes et al., 1999
). One way to circumvent these problems was the use of strong acylating agents, like heptafluorobutyric anhydride (HFBAA), resulting in high-mass compounds with a very poor adsorption on classical methyl-siloxane liquid phases (Zanetta et al., 1999
) and, consequently, that eluted at relatively low temperature. Furthermore, the derivatives present a very strong stability with time, the reaction can take place quantitatively even in the presence of salts, proteins, or other contaminants and the semi-acetalic group is not derivatized. This article reports the methodology of formation of their volatile derivatives and presents the GC/MS data obtained in the electron impact mode allowing the identification of different sialic acid derivatives found in various biological samples.
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Results and discussion |
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Because problems of stability with time were encountered for TMS derivatives, the same samples were analyzed at different periods of time after the addition of the acylating mixture (200 µl acetonitrile and 25 µl HFBAA) after evaporation of the diazomethane/ether mixture. Samples were immediately heated at 150°C for 5 min, evaporated under nitrogen, dissolved into dried acetonitrile, and analyzed. The results were compared to samples remaining for 1 month at room temperature in the acylating mixture, heated at 150°C for 5 min, evaporated under nitrogen, and analyzed. The two types of samples were indistinguishable. This indicated that standing at room temperature in the acylating mixture did not introduce degradation of the sialic acid derivatives. This was not the case when already analyzed samples (depleted of the acylating mixture) were standing for such a period of time, even in closed vials. The quantity of the initial sialic derivatives was decreased concomitantly with the appearance of additional peaks (including de-O-acetylated sialic acids). This indicated that O-acyl groups were relatively unstable when standing for a long period of time in a medium especially enriched in heptafluorobutyric acid, but remain stable in the presence of an excess of HFBAA. Nevertheless, already analyzed samples could be stored unaffected for at least 3 months after the addition of 25 µl of HFBAA.
GC separation of the HFB derivatives of sialic acids
Analyses of standard samples indicated that most of the HFB derivatives of the methyl esters of sialic acids could be separated using classical methyl-siloxane columns. As previously reported, HFB derivatives of sugars poorly interacted with these liquid phases (Zanetta et al., 1999). For example, the HFB derivatives of malto-tetraose (mass 3214 Da) is eluted before the C24:0 fatty acid methyl ester (mass 382 Da). Most of the HFB derivatives of the methyl esters of sialic acids were eluted between 140 and 200°C, depending on their mass and on their degree of initial O-substitution and the nature of their N-substitution. For example, the HFB derivative of the methyl ester of Neu5Ac (mass 1107 Da) was eluted (Table I) before that of Neu4,5Ac2 (mass 953 Da). The derivatives of the different mono-O-acetylated Neu5Ac were eluted before those of the di-O-acetylated Neu5Ac. The derivative of N-glycolyl neuraminic acid (Neu5Gc) (mass 1319 Da) was eluted at a temperature higher than that of Neu5Ac, whereas the derivative of Kdn (mass 1262 Da; without N-acyl group) was eluted before that of Neu5Ac (Table I and Figure 2b). The general rules of separation were that compounds having additional acetyl groups were retarded despite their lower mass, whereas compounds with a 9-O-lactyl group were eluted before the 9-O-acetyl compounds despite their higher mass (Table I).
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Identification of the parent members of the families of sialic acids
All sialic acids derivatives (except those derived from Kdn) gave an extremely predominant ß-anomer (96%) after acid hydrolysis. As shown in Figure 3a,b, Neu5Ac and Neu5Gc presented very different fragmentation patterns allowing their unambiguous identification in complex mixtures. Anomers of Kdn were found in a proportion close to 2/31/3. The proportion between the anomers changed slowly if samples were kept in the acylation mixtures for a long time because of mutarotation due to the presence of a free hydroxyl group on the C(2) carbon atom. As for Neu5Ac and Neu5Gc (not shown), the EI spectra of the HFB derivatives of Kdn anomers were significantly different (Figure 3c,d), the difference being concerned mostly with the intensities of the ions.
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It should be stressed that another series of ions were found derived from M 50/51 (corresponding to a loss of a fluorine/hydrofluoric acid and of a methanolate group [M F/HF OCH3]). This loss of mass is generally accompanied by an additional loss corresponding to a mixed anhydride between two neighboring O-acylated carbon atoms. For example, compounds having non-O-acylated C(9) and C(8) gave an ion at M 50410 (410 is the mass of [CF3CF2CF2CO]2O). Another loss of mass relative to M 50 was 256 for compounds having a single acetyl group on C(7)C(9) and 102 ([CH3CO]2O) for those having two acetyl groups on these carbon atoms. For compounds having an 8-O-sulfate group the same mechanism gave an intense positively charged ion at m/z 295 (Figure 4) corresponding to a charged mixed anhydride between heptafluorobutyric and sulfuric acid ([CF3CF2CF2CO-O-SO3H2]+). This series of ions was extremely important for confirming the position of the substituents outside the pyranic ring.
In the area of low masses, several ions were of fundamental interest for the identification of the different series of compounds. For example, a basic ion at m/z 81 was present, corresponding to the pyranic ring depleted of all substituents with a series of double bounds in resonance. An ion at m/z 238 was characteristic of Neu5Ac derivatives was already found to be characteristic of -amino-alcohols like long-chain bases (Pons et al., 2000
).
Mono- and multi-O-acylated derivatives of Neu5Ac
All mono-O-acetylated derivatives of Neu5Ac (mass 953 Da) could be identified through the intense doublet of ions at m/z 861/862 (Figure 5ad) derived from the L1 ion by the loss of an additional methyl group. The derivative of Neu4,5Ac2 (especially abundant in equine material) could be unambiguously identified throughout the different mono-O-acetylated Neu5Ac (Figure 5a) because of the presence of additional intense doublet of ions at m/z 704/705 (see Specificity of the fragmentation patterns of Neu5Ac, Neu5Gc, and Kdn derivatives) and 490/491 derived from the former by a loss a heptafluorobutyric acid group. The two other derivatives lacked this ion. Neu5,7Ac2 (Figure 5b) was identified through two ions at m/z 790. Neu5,9Ac2 (Figure 5c) was characterized by its intense ion at m/z 490, a medium intensity ion at m/z 530 (L1 [CH3CONH2 + C(9)H2OCOCH3 + CF3CF2CF2COOH]), its weak ion at 745 and overall by the ion at m/z 73 [C(9)H2OCOCH3]+ (Figure 4). Neu5,8Ac2, identified as a very minor constituent in bovine submandibular gland mucin (BSM), showed intense characteristic ions at m/z 492 and 307 (Figure 5d). These compounds showed significantly different RTs (Table I) and are separated from each other. Interferences in the interpretation of the spectra of O-acetylated derivatives of Neu5Gc could be solved because the corresponding ions showed a significantly lower mass (859/860 instead of 861/862). The reverse was observed with the O-acetylated derivatives of Kdn, because the second-order ions showed masses significantly higher (predominant ion at m/z 863). As shown in Table I, the relative retention times (RTr) to Neu5Ac provided an additional unambiguous confirmation of the nature of the compounds.
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Di-O-acetylated derivatives of Neu5Ac (mass 799 Da) gave a common L1 reporter ion at m/z 707/708. Unfortunately, such compounds were rare in our samples and only Neu5,7,9Ac3, Neu5,8,9Ac3, and Neu4,5,9Ac3 were identified (Figure 6ac). Neu5,7,9Ac3 was characterized by ions at m/z 73 and m/z 153 (ring depleted of substituents except the C(7) carbon atom) and m/z 379 (the latter plus the HFB derivative of the C(8) carbon atom). Neu5,8,9Ac3 was characterized by the presence of intense ions at m/z 145 (substituted C(9) and C(8)) and 103 (the latter minus CH2=C=O; Figure 4). Neu4,5,9Ac3 was characterized by the ion at m/z 73 (acetylated C(9)) and by the absence of the ion at m/z 238 simultaneous with the presence of the ion at m/z 84 (Figure 4).
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Highly acetylated derivatives of Neu5Ac were detected as very minor compounds in the mucins from the human saliva and in horse serum glycoproteins. They included the per-acetylated sialic acid Neu4,5,7,8,9Ac5 (Figure 7b) and Neu4,5,7,9Ac4 (Figure 7a).
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Three compounds possessing both the ion at m/z 227 (characteristic of the Neu5Gc family) and at m/z 112 (characteristic of the lactylated compounds) were detected (Figure 8). They included Neu8Ac5Gc9Lt (Figure 8a), Neu4Ac5Gc9Lt (Figure 8b), and the di-acetylated compounds Neu7,8Ac25Gc9Lt (Figure 8c) and Neu4,7Ac25Gc9Lt (Figure 8d).
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Intramolecular lactones of sialic acid derivatives
Substances, reproducibly present at variable intensities depending on the samples, were identified as lactones derived from different sialic acids but not from Kdn. These compounds lacked the molecular ion [M]+°, but had a series of weak ions at M 20 (M HF) and M 37 (M [F + H2O]). Series of ions were derived from M 18 by the additional loss of heptafluorobutyrate/and or acetate. These compounds lacked systematically the M 76/77 and/or M 91/92 ions characteristic of all sialic acids described above. This suggested that the carboxyl group of the C(1) carbon atom was not in the form of a methyl ester, but was involved in an intramolecular lactone. For the lactone derived from Neu5Ac (mass 879 Da), these ions corresponded to m/z 859, 842, and 814, respectively. An intense ion was derived from the M 18 ion by the loss of a heptafluorobutyric acid group (m/z 647 for the lactone of Neu5Ac). A weak but fundamental ion for the identification of these compounds was the ion at m/z 136, corresponding to the formation of a double ring (Figure 4). Based on these criteria, 1,7-lactones derived from Neu5Ac (Figure 11a), Neu5Gc (Figure 11b), Neu5,9Ac2 (Figure 11c), and Neu4,5,9Ac3 (Figure 11d) were identified (but not exclusively) in the eggs of Bufo bufo.
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8-O-methylated sialic acids
Several samples (including the mammalian mucin OSM) showed a weak peak with an RT in between Neu5Ac and Neu5,9Ac2 endowed with a typical fragmentation pattern, most of its intense ions being absent from the other compounds. Although the molecular ion [M]+° was absent for the most frequent compound, all fragment ions could be interpreted starting from a mass of 925 amu. From a low intensity L1 ion at m/z 848, a sequence of ions was observed corresponding to sequential losses of mass of 227 amu (C(9)H2OCOCF2CF2CF3), 44 amu (C(8)HOCH3), and 226 amu (C(7)HOCOCF2CF2CF3) characteristic of a O-methyl group on the C(8) carbon atom, the C(9) and C(7) being initially nonsubstituted. It was concluded that the compound was Neu5Ac8Me (Figure 12a). Such a compound was already described in lower organisms (Kamerling and Vliegenthart, 1982) and its presence in mammals was surprising, but it could not be an artifact due to the methodology. Indeed, treatment of standard Neu5Ac with the diazomethane reagent up to 15 days at room temperature did not produce Neu5Ac8Me. Furthermore, because the O-methyl groups are stable to the most common chemical procedures used for the liberation of monosaccharides from glycoconjugates (in contrast with N- and O-acyl groups), this compound should be distinguished from the bulk of other sialic acids (including Kdn) in classical monosaccharide analyses. This point was actually verified using GC/MS analyses of the HFB derivatives obtained after methanolysis (Zanetta et al., 1999
). The HFB derivative of the methyl ester of the O-methyl glycoside of the 8-O-methylated neuraminic acid (Neu) (eluted just before the major anomer of sialic acid) was actually detected, although at a low level (2% of sialic acids) in OSM, the mucin in which it was present at the same level. In the methanolysis products of other samples, this compound was absent or present at an extremely low level (0.01% of the total sialic acids) in agreement with the data obtained in the procedure reported here. Neu5,9Ac28Me (Figure 12b) was identified in the sialic acids of the skin of Anguilla anguilla. The two compounds were characterized by an ion at m/z 578 and 424, respectively, the former corresponding to the pyranic ring depleted from all its substituents except the C(7)C(9) substituted chain. The reason such a compound was not detected before in higher animals might be its extremely low level.
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The reason that sulfated compounds were volatile remained puzzling, but it was unlikely that the methyl ester of the sulfate group formed using diazomethane methylesterification would resist to the acylation procedure. The actual reason could be a protective effect of the HFB groups on the C(7) and C(9) carbon atoms against formation of hydrogen bonds between the sulfate group and oxygen atoms of the liquid phase of the capillary column. The fact that only three compounds were detected (Neu5Ac8S, Neu5Gc8S, and Neu4,5Ac28S) that all had HFB groups on the C(7) and C(9) might be indicative of this protective effect, the other unprotected compounds being not volatile or interacting too strongly with the liquid phase. This assumption was sustained by the observation that the use of shorter chain fluorinated acylating agents, such as trifluoroacetic anhydride or pentafluoropropionic anhydride, could not allow the detection the previous 8-sulfated compounds.
Presence of Neu
A compound (representing 5% of Neu5Ac, the major sialic acid of OSM) was eluted at an RT between those of Neu5Ac and Neu5,9Ac2 (Figure 13c). It did not present the ions at 238 and at 227 characteristic of Neu5Ac and Neu5Gc, respectively. The two anomers (the major represented 99.5 %; Figure 13c), showed an intense ion at m/z 505, due to the pyranic ring substituted with two HFB groups. This indicated that the amino group was initially not substituted. Consequently, this compound corresponded to Neu, the amino group on the C(5) carbon atom being initially free and, consequently, transformed into the N-HFB derivative. Such a compound has not been so far identified as a free molecule, although indirect evidence was provided immunologically for its presence in gangliosides associated with cancer (Sjöberg et al., 1995). The possibility that it was a degradation product was unlikely, since this compound co-existed with O-acylated compounds, which are much less stable than N-acylated compounds. The explanation why Neu can be identified using the present method is that the heptafluorobutyric anhydride acylation is able to disrupt the Schiff base spontaneously formed between the amino group on the C(5) and the keto group on the C(2) of Neu liberated on hydrolysis, progressively and definitely blocking the amino group as its HFB derivatives.
Sialic acids with a second amino group
Several extremely minor compounds with characteristic intense ions at m/z 703, 518, 304, and 276 were specifically detected in one of the BSM preparations analyzed in this study. These compounds also had the particularity of having both the ions at 238 and 227 characteristic of derivatives of Neu5Ac and Neu5Gc, respectively. The first series of ions was 1 amu lower than for the other compounds, suggesting the presence of a second amino group in the molecule. The ion at 227 indicated the presence of 5-glycolyl groups, whereas the ion at 238 suggested a N-acetyl group in another position vicinal to a hydroxyl group substituted by a HFB group. Based on the fragmentation patterns, it was deduced that these compounds were derivatives of Neu5Gc having an N-acetyl group on the C(7) carbon atom. These compounds were Neu9Ac7Am5Gc and Neu4,9Ac27Am5Gc (Figure 12cd), in which Am represented an acetamido group. They were likely due to a bacterial contamination of this particular BSM preparation. Indeed, similar compounds were previously identified in somatic antigens of Pseudomonas aeruginosa (Knirel et al., 1987, 1997), although the previous compound (5,7-di-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid; pseudaminic acid) did not contain an N-glycolyl group and O-acetyl groups were not detected.
Analysis on crude samples
This method could be applied successfully to very crude samples, like crude mucins from the eggs of frogs (or a single egg as the starting material and less than 1% injected onto the GC/MS apparatus), total saliva proteins, and so on. Because HFB derivatives are stable, the analysis could be performed without elimination of the protein or lipid part of the sample after hydrolysis. Because no purification of the liberated sialic acids is needed, a complete analysis can be performed starting routinely from nanogram amounts of total sialic acids. Major contaminants already observed were oligosaccharides derived from glucose present in the samples, but these constituents could be easily identified through their intense L1 ion at 947 (Figure 1b). This was true not only for the major constituents but also for compounds representing a few percent of the major compounds. For example, the area between RT 21.50 min and RT 24.00 min in Figure 1b showed extremely weak peaks, some of them being other sialic acids that could be identified together with contaminant oligosaccharides and phthalates. Fatty acid methyl esters or sphingoid bases derived from glycosphingolipids did not interfere with the sialic acid determination because: (1) the acetamido bonds (like the other bonds) were not cleaved significantly on the hydrolysis conditions used for sialic acid determinations; and (2) fatty acid methyl esters, when present, are, in majority, eluted after the area of separation of sialic acid derivatives.
Major contaminants actually observed were derived from phthalates. Consequently, plastic vessels were not recommended in the handling of samples for these analyses. Nevertheless, these compounds could be easily identified by their ions at 149 and the absence of ions at 69, 119, and 169, in such a way that they did not significantly interfere with the identification of the sialic acids. The other major interferences were due to methyl-siloxane compounds derived from the liquid phase but also from residues of previous analyses of TMS derivatives, intense ions at 73, but revealed by the presence of an intense ion at m/z 225, absent from HFB derivatives.
Although the present method allows the identification of most compounds, confirmations of the structures can be obtained through a complete monosaccharide analysis after cleavage of the glycosidic bonds by acid-catalyzed methanolysis followed by GC/MS analysis of the compounds as HFB derivatives (Zanetta et al., 1999). Such analyses could be performed on the same sample after analyses of the sialic acids. This allows detecting the presence of Neu (produced from Neu5Ac and Neu5Gc and of their O-acylated derivatives), Kdn derivatives, and O-alkylated derivatives. Additional confirmations can be obtained using a previous alkaline elimination of O-acyl groups (NaOH 0.1 M for 2 h at 37°C followed by neutralization with hydrochloric acid, evaporation, and formation of volatile derivatives). This allows identifying the disappearing peaks as O-acylated derivatives.
Conclusions and perspectives
This method presents important advantages compared to previously described techniques, essentially due to the stability of the HFB derivatives and the absence of derivatization of the semi-acetalic group with HFBAA. In contrast with other methods, it does not need intermediate steps of purification of sialic acids after the mild hydrolysis step. For glycolipids or oligosaccharides, the samples can be analyzed in the same vial, without elimination of the other derivatives. For glycoprotein analysis, a short-term centrifugation step is needed to eliminate the insoluble material from material with a very low sialic acid content. In fact, in most cases, the centrifugation step can be avoided, but cleaning of the GC injector should be performed frequently. Although soluble peptides and amino acids are still present, they do not interfere with the analysis. This type of contamination cannot be easily solved using shorter fluorinated anhydrides such as trifluoroacetic anhydride (Zanetta et al., 1972) and pentafluoropropionic anhydride (unpublished data).
From the MS analysis, HFB derivatives of sialic acids present the advantage of having specific ions that allow the immediate selection of these compounds in very impure samples by chromatogram reconstitution using the ion at m/z 169, characteristic of HFB derivatives. Furthermore, the ions of HFB derivatives are generally not saturating relative to the higher-mass ions, and it is possible to record spectra for all ions above 45 amu. This appears to be a significant point because important ions for the identification of some compounds have relatively low masses (ion at m/z 73 for 9-O-Ac, 84 for 4-O-Ac [except Neu4,5Ac2], 112 for 9-O-lactyl, 122 for 8-O-sulfate, etc.). Furthermore, the modalities of fragmentation of these derivatives allow a relatively easy structural assignment for most compounds.
This method is by far more sensitive than others. The first important point is that it does not need a purification step of the liberated sialic acids, which may lead to loss of sialic acids and to isomerization of O-acetyl groups. Indeed, classical techniques used a dialysis of the hydrolyzed mixture to recover sialic acids in the dialysate, followed by ion exchange chromatography. This procedure needs relatively high quantities of sialic acids (Schauer and Kamerling, 1997), even if the following analysis steps are very sensitive and specific. Routinely, 0.11 ng total sialic acids are injected onto the GC/MS apparatus to avoid saturation of the TIC detector. The second important point is the wide range of detection of specific ions and/or complete mass spectra (1/1000) of the MS detectors. Sialic acids present at a concentration less than 1/1000 relative to the major peaks can be unambiguously identified. Most of the extremely minor TIC peaks (most of them appear as definite peaks only examining areas not containing the major peaks) give spectra allowing the identification of these compounds.
Because purification of the liberated sialic acids (for review, see references in Schauer and Kamerling, 1997) is not needed, the method allows identifying compounds that do not respond to the expected properties of sialic acids, that is, monosaccharides with a single carboxylic acid negative charge. The presence of 1,7 lactones has been probably missed because of the absence of a negative charge in these compounds. Such compounds cannot be revealed using the reagents specific for 2-keto-carboxylic acids but appear as the corresponding sialic acid after alkaline treatment. Furthermore, lactones may have been lost during ion exchange chromatography. Similarly, Neu was not detectable because of its transformation into a Schiff base. Sulfated sialic acids should have been also missed during ion-exchange chromatography because of the strong acidic charges of these molecules. Although we expect that multi-O-acetylated 8-O-sulfated sialic acids will not be volatile, this technique allowed to clearly identifying Neu5Ac8S, Neu5Gc8S, and Neu4,5Ac28S in routine analyses.
Because of its simplicity and sensitivity, this method allows routine analyses on minute quantities of material, for example, a single frog egg. It will be possible to analyze directly human samples to evaluate if qualitative and quantitative modifications of sialic acids occur in different pathologies, especially cancer, and to analyze in different tissues the presence of putative ligands of bacterial or viral agglutinins. But it could also open a wide field of research in the field of biochemistry because it could reveal the presence of unexpected sialic acids in different organisms and tissues. This could be the basis of specific searches of new enzymatic activities, needed for the synthesis and degradation of these compounds, as already discovered in several systems. This could be also the basis for the study of new molecular and biological interactions in the function of fundamental physiological mechanisms, still unexpected because of the absence of knowledge on the existence of strange sialic acid derivatives. One recent example was the identification of the high affinity ligand of human interleukin 4, the Neu5Ac1,7-lactone (Cebo et al., 2001), a compound for the first time detected at the surface of human leukocyte using the present methodology.
The finding of new sialic acid species and the occurrence of rare types like 8-O-methylated or 8-O-sulfated, known so far only in echinoderms (Bergwerff et al., 1992; Schauer and Kamerling, 1997
; Schauer, 2000
), in animals higher than these, even in mammals, is a challenge for enzymologists and cell biologists, to search for the responsible enzymes and genes on the one hand and for the cell biological significance on the other. This is also valid for the sialic acid lactones identified and for the nonsubstituted Neu molecule itself. In addition to the ongoing research on the metabolism of O-acetylated sialic acids in mammals, the biosynthesis of the various O-acetylated Kdn derivatives found in amphibian needs elucidation.
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Materials and methods |
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Gas-liquid chromatography and MS
For GC/MS analysis, the gas-liquid chromatography separation was performed on a Carlo Erba GC 8000 gas chromatograph equipped with a 25 m x 0.32 mm CP-Sil5 CB low bleed/MS capillary column, 0.25 µm film phase (Chrompack France, Les Ullis, France). The temperature of the Ross injector was 260°C, and the samples were analyzed using the following temperature program: 90°C for 3 min then 5°C/min until 260°C. The column was coupled to a Finnigan Automass II mass spectrometer (mass detection limit 1000) or, for mass larger than 1000, to a Riber 10-10H mass spectrometer (mass detection limit 2000). The analyses were performed routinely in the EI mode (ionization energy 70 eV; source temperature 150°C). When necessary, detection was performed in the chemical ionization mode in the presence of ammonia (ionization energy 150 eV, source temperature 100°C). The chemical ionization detection was performed for positive ions or negative ions, in separated experiments, the latter allowing the quite specific detection of HFB derivatives with a higher sensitivity (Zanetta et al., 1999).
Cleavage of sialic acid residues from glycoproteins and glycolipids and preparation of the samples for derivatization
All experiments were performed in heavy walled Pyrex tubes with a Teflon-lined screw cap. In preliminary experiments, sialic acids were liberated from glycolipids and from glycoproteins using two different techniques: diluted formic acid at pH 2.0 for 90 min at 80°C, or 2 M acetic acid for 90 min at 80°C. Because of similar cleavage yields and of the easier elimination under vacuum of acetic acid relative to formic acid, the former was used in all experiments.
The tubes containing the glycoprotein or glycolipid samples were evaporated under vacuum at room temperature then supplemented with 2 M acetic acid (at least 2 ml/mg protein or 2 ml/100 µg glycolipid, with a maximum of 500 µl per reaction vial). The tightly closed vials were gently shaken and placed in an oven at 80°C. After 90 min, the samples were cooled, and after addition of two drops of toluene, were evaporated to dryness using a rotary evaporator (with a Teflon-lined adaptation to the reaction vessels) at room temperature. They were treated differently depending on their nature and on their content in sialic acids:
(a) For most mucins and sialylated glycolipids containing high levels of sialic acids, the dried samples were directly derivatized and analyzed without additional purification.
(b) For protein fractions containing very low levels of sialic acids, the evaporated hydrolyzed samples were dissolved into 1 ml water and left for 1 h at 4°C. The precipitate was eliminated by centrifugation at 4°C for 30 min at 4000 rpm, and the supernatant was evaporated again under vacuum at room temperature. The dry sample was submitted to derivatization.
(c) For total cell homogenates or samples containing extremely low amounts of sialic acids and glycoproteins showing a very weak precipitation, the samples were treated as above (b) and resuspended in a small volume of water (100 µl/mg initial protein). It was passed through a small column of CM-Trisacryl (200 µl of swollen gel in a Pasteur pipette closed with glass wool). The run through of the column and 500 µl additional water eluate were recovered and evaporated with a rotary evaporator as above, then derivatized as below.
Derivatization of sialic acids
The dry samples (0.11 µg of total sialic acid) were supplemented with 100200 µl of anhydrous methanol (Kamerling and Vliegenthart, 1982; Fontaine et al., 1994
) at the bottom of the vial (to partially dissolve sialic acids), followed by the addition of 200 µl of a diazomethane solution in ether, and the tubes were tightly closed. Diazomethane was prepared in a WheatonTM apparatus according to the procedure proposed by the manufacturer, and the diazomethane solution was kept as aliquots in tubes identical to the reaction vials at room temperature in a ventilated hood (diazomethane is a strong irritatant and carcinogenic agent; consequently drastic cares have to be taken during handling). 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. 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 the required volume of acetonitrile previously dried on calcinated calcium chloride (Zanetta et al., 1999
), and aliquots were injected onto the Ross injector of the GC/MS apparatus. Optimal analyses were obtained injecting 0.11 ng of each sialic acid derivatives in the GC/MS apparatus, but compounds could be identified safely at the picogram level. If analyses are performed on higher amounts of material, the quantities of the reagents have to be increased in proportion.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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