CGL Heptafluorobutyrate Derivatives Glycosides

1999 Oxford University Press

Glycobiology vol. 9 no. 3 pp. 255266, 1999

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Gas-liquid chromatography of the heptafluorobutyrate derivatives of theO-methyl-glycosides on capillary columns: a method for the quantitativedetermination of the monosaccharide composition of glycoproteins and glycolipids

Jean-Pierre Zanetta1, Philippe Timmerman and Yves LeroyLaboratoire de Chimie Biologique, CNRS UMR 111, 59655 VilleneuvedAscq Cedex, France

Received on May 18, 1998; revised on July 22, 1998; accepted on July 27,1998

1To whom correspondence should be addressed at: Laboratoire de ChimieBiologique USTL, CNRS UMR 111, 59655 Villeneuve dAscq Cedex,France

We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberatedfrom glycoproteins and glycolipids following methanolysis.The stable derivatives of the most common monosaccharidesof these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc,GlcNAc, GalNAc, Neu5Ac, KDN) can be separated andquantitatively and reproducibly determined with a highdegree of sensitivity level (down to 25 pmol) in the presenceof lysine as an internal standard. The GlcNAc residue boundto Asn in N-glycans is quantitatively recovered as two peaks.The latter were easily distinguished from the other GlcNAcresidues of N-glycans, thus allowing a considerable improve-ment of the data on structure of N-glycans obtained from asingle carbohydrate analysis. The most common contamin-ants present in buffers commonly used for the isolation ofsoluble or membrane-bound glycoproteins (SDS, TritonX-100, DOC, TRIS, glycine, and polyacrylamide or salts, aswell as monosaccharide constituents of proteoglycans ordegradation products of nucleic acids) do not interfere withthese determinations. A carbohydrate analysis of glycoproteinsisolated from a SDS/PAGE gel or from PDVF membranes canbe performed on microgram amounts without significantinterferences. Since fatty acid methyl esters and sphingosinederivatives are separated from the monosaccharide peaks,the complete composition of gangliosides can be achieved ina single step starting from less than 1 g of the initial com-pound purified by preparative Silicagel TLC. Using electronimpact ionization mass spectrometry, reporter ions for thedifferent classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, andKDN) allow the identification of these compounds in verycomplex mixtures. The mass of each compound can be deter-mined in the chemical ionization mode and detection of positiveor negative ions. This method presents a considerableimprovement compared to those using TMS derivatives. In-deed the heptafluorobutyrate derivatives are stable, andacylation of amino groups is complete. Moreover, there is nointerference with contaminants and the separation betweenfatty acid methyl-esters and O-methyl glycosides is achieved.

Key words: GLC/GC/MS, mass spectrometry/monosaccharide

IntroductionThe interest over the carbohydrate moieties of glycoconjugates hasbeen increasing because of their postulated functions in cell adhesionand recognition mechanisms and their importance in signaltransduction and as markers of differentiation and carcinogenesis.The determination of the monosaccharide composition is a key stepin choosing the strategy for glycan analysis. The most commonmethod is the capillary column GLC analysis of the trimethylsilylderivatives (TMS) obtained from O-methyl-glycosides after metha-nolysis induced cleavage of glycosidic bonds (for review, seeChaplin, 1994). This cleavage procedure, when used in controlledconditions (and especially the use of anhydrous methanol/HClreagent; Zanetta et al., 1972), allows the almost quantitativeliberation of the glycoprotein and glycolipid monosaccharides asstable O-methyl-glycosides. However, an intermediate step ofre-acetylation of these compounds is necessary before the formationof the TMS derivatives in the classical procedure (for review, seeChaplin, 1994). Unfortunately, this re-acetylation step is timeconsuming, and the reaction yield is particularly difficult to control.As a result, the molar ratio of N-acetyl-hexosamines and sialic acidfluctuates from an experiment to the other, rendering mono-saccharide molar composition of a glycan often problematic.

One way to circumvent such a difficulty is the formation ofvolatile derivatives of O-methyl-glycosides using a strongacylating agent (acid anhydride). In a previous paper (Zanetta et al.,1972), the most common constituents of glycoproteins andglycolipids (and all constituents of glycolipids) could be determinedquantitatively as trifluoroacetate derivatives (TFA) using aseparation by classical GLC. However, this method lacked thesensitivity required for applications to small amounts of material(less than 100 g of glycoprotein). The reason was that the use ofcapillary columns was prevented by the presence of an excess oftrifluoroacetic anhydride which destroyed the liquid phase.Because of the volatile nature of the TFA derivatives, the excessof anhydride could not be eliminated without the complete lossof the most volatile compounds. We hypothesized that one wayto solve this problem was to produce less volatile derivativesusing an acylation with anhydrides of higher mass (pentafluoro-propionic or heptafluorobutyric anhydrides). This article demon-strates that quantitative monosaccharide analyses of glycoproteinsand glycolipids can be achieved on capillary columns using theheptafluorobutyrate derivatives of the O-methyl-glycosides.

Results and discussionChoice of the acylating agentStandard monosaccharide mixtures were submitted to methanolysis(see Materials and methods), followed by acylation withpentafluoropropionic or heptafluorobutyric anhydrides, respect-ively. The derivatives were injected into the SE-30 column, andthe area of the different peaks was quantified. These areas werecompared to those of the same compounds injected after thecomplete evaporation of the acylation mixture under a stream of

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nitrogen and resolubilization in dried acetonitrile. Pentafluoro-propionate derivatives of fucose, arabinose, and xylose and to alesser extent the hexose derivatives showed a significant decreaseduring evaporation (a maximum of 20% loss for the most volatilecompounds). In contrast, identical areas were found for allheptafluorobutyrate derivatives, even working with less than1 nmol of each monosaccharide in the reaction vessel. Thisadequate volatility was verified after injection on capillarycolumns, the surface of the peaks being constant after evaporationof the solvent in the Ross injector.

Conditions of acylationThe acylation reaction with HFBAA was performed in acetonitrilebecause this solvent was able to solubilize the O-methyl-glyco-sides formed during methanolysis and because it formed a singlephase with HBFAA (in contrast with less polar solvents such asdichloromethane, chloroform, heptane, methyl or ethyl acetate).Kinetics of acylation of the different O-methyl-glycosides werefollowed either at room temperature or at 100C or 150C,analysing the areas of the different peaks of standard mixtures.When experiments were performed at room temperature, theacylation was complete only after 24 h, the acylation being slowerfor monosaccharides possessing amino groups (hexosamines andsialic acid). When acylation was performed at 100C, theacylation was maximal after 30 min; the relative molar responsesdid not vary for longer times of heating. At 150C, the acylationwas complete within 5 min (this procedure being recommendedsince the reaction takes place as a reflux, also acylating thematerial remaining on the vessel wall).

Because acetonitrile showed some trailing on classical columns,we tried to solubilize the evaporated HFB derivatives in othersolvents, i.e., heptane or dichloromethane. Heptane provoked animportant loss of most compounds (especially hexosamines andsialic acid, but also pentoses and hexoses) at all initial concentra-tions of monosaccharides because HFB derivatives were notsoluble. A significant loss of the same compounds was observedwith dichloromethane when the initial concentration of themonosaccharide was more than 5 nmol in the vessels. In contrast,acetonitrile completely dissolved the different compounds andappeared suitable for capillary column analysis.

Stability of the heptafluorobutyrate derivativesAfter acylation, the HFB-derivatives kept at room temperature in theclosed reaction vial were analyzed at different periods of time. Novariations were observed during several months, as already observedfor the trifluoroacetate derivatives (Zanetta et al., 1972). The sametype of experiment was also performed on the HFB derivatives

stored for different periods of times in dried acetonitrile after thecomplete evaporation of the acylation mixture. No variations wereobserved when samples were stored for 2 days in the closed vial withthe exception of the derivatives corresponding to the GlcNAcresidue involved in the N-glycosidic bond (see below). Repetitive(up to 10 times) opening of the vials for time to time injections didnot modify significantly the relative molar responses of the differentcompounds with the exception of the previous derivatives and,specifically, the derivatives of the furanic forms of all compounds(Fuc, Gal, GalNAc, GlcA, and GalA) and the derivatives of sialicacid and KDN. However, the initial response of these compoundscould be restored, adding 25 l of HFBAA and heating for 5 minat 100C. This indicated that the HFB derivatives presented a strongstability in neutral or acidic conditions. Because of this stability, thecleaning of the reaction vessels before reuse should first involve animmersion in an alkaline solution (1 M NaOH for 10 min). Tubeswere cleaned thereafter by heating for 1 h at 80C in 50% sulfuricacid.

Choice of GLC separationsFrom previous observations (Zanetta et al., 1972, 1973), it appearedthat fluorinated compounds strongly interact with fluorosiliconeliquid phases and are poorly adsorbed on classical silicone phases,in such a way that they are eluted from the columns at temperaturelower than their boiling points. In contrast, compounds rich inmethylene groups strongly interact with classical silicone phases. Infact, despite their high mass, HFB of O-methyl-glycosides wereeluted before fatty acid methyl-esters (FAMEs).

The chosen chromatographic conditions were: injector anddetector temperature 260 C; helium carrier gas 0.8 bar;temperature program: 100C to 140C at 1.2C/min, followedby 4C/min until 240C (when samples contained only mono-saccharide derivatives, the temperature program was stoppedafter the elution of the neuraminic acid peak). Using theseconditions, almost all HFB of the isomers of O-methyl-glyco-sides (Xyl, Ara, Rha, Fuc, Gal, Man, Glc, GlcNAc, GalNAc,NeuAc, and KDN) were separated from each other (Figure 1,Table I). The single overlapping was observed for the -mannoseisomer and a furanic form of GalNAc. Furthermore, FAMEs wereeluted at higher retention time than the derivatives of NeuAc. Thequality of the separations remained stable during at least sixmonths of intense use (about 600 injections), indicating that thederivatives did not destroyed the columns. However, when HFBderivatives are chromatographed on a column on which otherderivatives were analyzed (acetates or TMS derivatives), theseparation of the second isomer of Glc and the third of Gal areonly partially achieved. This partial overlapping disappearedafter two to three runs of HFB derivatives.

Fig. 1. Gas chromatography of the heptafluorobutyrates of O-methyl glycosides on a capillary column. (a) GC chromatogram of a mixture of standardmonosaccharides (Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac) and fatty acids (C16, C18, C20, C22 as major compounds) submitted to methanolysis thenacylation by HFBAA. Note that all constituents of N-glycans are separated the one from the other and completely separated from FAME. The -isomer of Glc isonly partially resolved from a galactose isomer due to the injection of HFB derivatives immediately after a chromatography of TMS derivatives. Inset: separationof the different isomers of O-methyl glycosides of pentoses and deoxy-hexoses (Ara, Rha, Xyl, and Rib). The lines inside the chromatogram represent thetemperature program. (b) GC chromatogram of glycoproteins found in wasps net. Note the complete separation of Ara, Rha, Fuc, the presence of two peaks(GN1) corresponding to the first GlcNAc residue of N-glycans. This chromatogram revealed the presence of small amounts of glucuronic acid (peak in-betweenthe major peaks of Gal and Man, and the two peaks migrating between the two first peaks of GalNAc; Rt = 25.43, 31.95, and 32.39 min, respectively). (c) GCchromatogram of the mucins of the eggs of Pleurodeles walti. This materiel contained high amounts of KDN, perfectly separated from all other compounds. Thecarbohydrate composition deduced from this analysis performed starting from 10 g of mucin was identical (less than 1% variation) to a previous analysisperformed on 1 mg mucin using the TFA derivatives (Zanetta et al., 1972). Because these material were injected onto a column perturbed by the injection ofacetylated compounds, the separation of the Glc and Gal isomers was not optimal.

HFB derivatives of O-methyl glycosides

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Table I. Retention time and proportions of the different isomers of theheptafluorobutyrates of O-methyl-glycosides (the Rt are reproducible within0.02% of min)

Compounds Retention time % of totalMeso 23.09 100.00Manni 15.19 100.00Xyl 16.01 66.48Xyl 16.32 33.52Ara 11.75 16.82Ara 14.91 83.18Rha 14.55 94.48Rha 17.70 5.52Rib 13.83 14.76Rib 16.83 15.38Rib 18.66 69.86Fuc 13.43 12.04Fuc 14.43 5.96Fuc 16.73 55.23Fuc 18.94 26.67Gal 20.04 19.75Gal 22.79 6.18Gal 25.37 49.38Gal 27.46 24.69Man 26.41 93.05Man 29.49 6.95Glc 26.89 67.49Glc 27.27 32.51GlcNa 20.17 48.90GlcNa 22.29 51.10GlcN 34.98 6.67GlcN 35.90 83.83GlcN 36.56 9.50GalN 29.41 18.13GalN 32.65 24.30GalN 35.48 52.59GalN 36.72 4.98Neu5 42.21 11.83Neu5 43.80 88.17KDN 38.97 27.78KDN 39.72 72.22GlcN-OH 16.32 100.00GalN-OH 20.63 100.00GlcA 25.43 29.63GlcA 31.95 17.32GlcA 32.39 53.05GalA 21.12 41.71GalA 22.91 13.76GalA 31.51 36.46GalA 33.61 8.07ManNb 34.64 34.49ManNb 35.77 42.69ManNb 36.85 19.37Asn 11.09 100.00Lys 38.65 100.00

aThe two peaks correspond to the derivatives formed during the methanolysisof the glycosylamine of GlcNAc involved in the N-glycosidic bond ( and isomers of glucosamine).bThe derivatives of N-acetyl-mannosamine interfere with the separation ofthose of GlcNAc and GalNAc, but this compound has not been so faridentified in glycoproteins or glycolipids.

Choice of an appropriate internal standard

Classical techniques used polyols as internal standard (such asmeso-inositol or mannitol). In fact mannitol could be released byreductive -elimination procedure from glycans with an O-linkedmannose residue (Chiba et al., 1997), whereas meso-inositol is anessential constituent of GPI-anchors. Moreover, in the presentsystem, meso-inositol showed trailing and interfered with thesecond Gal isomer. Because of these possible interferences, wepreferred amino acids, the carboxyl group being methyl-esterifiedduring methanolysis and the amino group acylated with HFBAA.Throughout the different amino acids presenting stable heptafluoro-butyrate derivatives (Zanetta and Vincendon, 1973), lysine(Rt = 38.65) showed a chromatographic behavior compatiblewith the separation of the classical monosaccharides of glyco-proteins and of glycolipids and did not interfere with anymonosaccharides. Because of the presence of two minor impuritiesin commercially available lysine, it was necessary to recrystallizeit from water. Subsequent studies of the possible contaminationsof glycolipid and glycoprotein samples (see below) indicated thatlysine was a suitable internal standard.

Determinations of the relative molar responses

The relative molar responses (RMR) of the different HFBderivatives were first determined on equimolar mixtures ofdifferent monosaccharides submitted to methanolysis in thestandard conditions, followed by acylation with HFBAA andanalyzed on the two types of GLC columns. Because of thepresence of significant amounts of impurities in commerciallyavailable standards, these RMR were only approximate. Correctionsdue to the presence of the impurities eliminated some divergencesobserved between the different compounds of the same mass.These RMR were compared to those obtained from the analysisof oligosaccharides, reduced oligosaccharides or glycoaspara-gines, the structure and purity has been previously determined bymethylation and NMR analysis. The final data from thesedeterminations are presented in Table II: (1) taking into accountsall isomers of the different monosaccharides (RMR); (2) takinginto account the major isomer (RMRmp), since the proportionsof the different isomers of each monosaccharides were constant(with the exception of the derivative of sialic acid and that ofgalactose). Therefore, the quantitation of an analysis could beperformed into two steps: (1) verification that the proportions ofthe different isomers are correct (a point that indicated theabsence of contaminant in the major peaks); (2) calculation of thearea of each monosaccharide based on the RMR of the major peak(RMRmp; Table II). The late form of calculation could not beapplied systematically to the NeuNAc derivatives because thesescompound are eluted, depending on the sample, either as a singlepeak or as two peaks. In the latter case, the major anomerrepresented always 88.17%, a point that remains to be explained.Similarly, the integration of the two peaks corresponding to thederivatives of the major product issued from the GlcNAc residueinvolved in the N-glycosidic bond, i.e., glucosamine (Maes et al.,1999), was needed, since these proportions were varying withtime, due to a slow change in the proportions of the and isomers from a methanol/HCl to an acetonitrile medium. The caseof the response of Gal will be discussed below.


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Table II. Relative molar response (RMR) of the heptafluorobutyrates ofO-methyl-glycosides and relative molar response relative of the major isomerof each compound (RMRmp)

Compounds RMR/IS RMRmp/IS (only the(all peaks integrated) major peak integrated)

Meso 1.000 1.000GalNac-OH 0.850 0.850GlcNac-OH 0.850 0.850Xyl 0.850 0.565Ara 0.750 0.624Rha 0.900 0.850Fuc 0.896 0.495Gal 0.755e 0.559eMan 0.930 0.865Glc 0.930 0.628GlcNAc 0.872 0.731GalNAc 0.751 0.395Neu5Ac 1.000 0.882bKDN 1.000 0.722a

GlcAd 0.950 0.504GalAd 0.930 0.388GlcNc 0.750Asn 0.500 0.5002Lys (IS) 1.000 1.000

The RMR (0.2% of the indicated values) are mean of at least 30 differentdeterminations both on standard of monosaccharides submitted tomethanolysis then acylation with HFBAA and on glycoproteins, glycolipidsor oligosaccharides and glycoasparagines of known compositions.aTheoretical value, deduced from the RMR of the derivatives of Neu5Ac.bIn some glycoconjugates and on the standard Neu5Ac, two peaks wereobserved; the RMR = 0.882 correspond to that of the major peak of NeuAc.In analyses where Neu5Ac gave a single peak, the RMR = 1.000 of NeuNAcwas applied.cBecause of the variations in the relative proportions of the two peaks, theRMR correspond to the result of the sum of their area.dNot released significantly from glycosaminoglycans during methanolysis(115% depending on the compounds), but could be partially released frombranched oligosaccharides. For simplifying the calculations of thecarbohydrate content of glycoproteins or glycolipids, the addition of 2 g ofLys as the internal standard was considered as 1 g of internal standard (IS),since the relative molar response of Lys is only half of that of many O-methylglycosides.eWhen O-glycans are present (presence of GalNAc or GalNAc-OH, thesurface of all galactose peaks should be integrated since the proportion of thefirst isomer (Rt = 20.04) is significantly increased. The RMRmp allows arapid calculation of the molar ratio since calculation is based only on the areaof the major isomer of each monosaccharide.

The analysis of the different glycoasparagines and of glyco-proteins of known monosaccharide compositions indicated adeficit of GlcNAc, when compared to the oligosaccharides witha single or two GlcNAc residues. However, two major extraneouspeaks were detected for glycoproteins containing N-glycans atRt = 20.17 and 22.29 min, respectively (see Figures 1c, 2). A thirdextraneous peak was observed for glycoasparagines (Rt =11.09 min), that corresponded to the di-methyl-ester N-heptafluoro-butyrate of aspartic acid by EI/MS (357) and CI/MS (M + 18 = 375).Since this compound was a little bit too volatile (Zanetta andVincendon, 1973), it could not be exactly quantified using theevaporation needed for capillary column analysis. Withoutevaporation, and taking into account a RMR of 0.500 for thisderivative of Asp, it could be calculated that it corresponded toone residue of Asp per glycoasparagine. This indicated that theN-glycosidic bond was almost quantitatively cleaved (Maes et al.,

1999). The nature of the products liberated from GlcNAc-Asn bymethanolysis will be reported in detail elsewhere (Maes et al.,1999). From the analysis of different N-glycoproteins andglycoasparagines, a RMR of 0.750 was assigned to the sum ofthese two compounds. Therefore, for the analysis of glycoproteinscontaining N-glycans, the ratio of the different monosaccharideswas calculated relative to these constituents representative of theGlcNAc residue involved in the N-glycosidic bond.

Table III. Retention times of fatty acid methyl esters and of sphingosinederivatives obtained after methanolysis and acylation with HFBAA

FAME Retention RMR/IS Sphingosine Retention RMR/ISatime time

C16:0 48.98 1.620 C18:1 45.81 1.560C18:0 54.55 1.800 C18:1 48.13 1.560C18:1 53.92 1.790 C20:1 58.45 1.900C18:2 53.76 1.780 C20:1 57.43 1.900C20:0 58.65 2.300 C20:1 58.89 1.900C20:3 57.97 2.280C20:4 57.47 2.250C22:4 62.72 2.560C22:6 62.35 2.540C24:0 65.34 2.830

A more complete study of the fatty acid methyl esters and of the HFBderivatives of hydroxylated FAMEs will be published elsewhere.aThe RMR of the different sphingosines were determined from the dataobtained on the GM1 gangliosides, assuming that the response wasproportional to the number of carbon atoms. For FAMEs and sphingosine adifference of Rt of 0.20 min resulted in a complete separation of the two peaks.

Applications to glycoprotein and glycolipids

Complete composition of gangliosides. HFB derivatives ofO-methyl-glycosides were completely separated from FAMEsand from the HFB derivatives of sphingosines on the capillarycolumn. The C14:0 FAME was eluted later than the NeuAc HFBderivative. The different FAME were fully separated from eachand are eluted before 240C (Figure 1a). Because the derivativesof sphingosines presented a high mass, they were eluted inbetween fatty acids (Figure 2). Therefore, it was possible todetermine simultaneously the carbohydrate, sphingosine andfatty acid composition of gangliosides without interference witheach other. The compositions of the GM1 and G5b gangliosidesare given in Table III. The determination of the nature of the fattyacid methyl esters was obtained by comparison with standardFAMEs and GC/MS analysis. The nature of the heptafluorobutyratederivatives of sphingosines was given by GC/MS analysis(Figure 2). Using EI ionization, the different bases gavecharacteristic fragments at M = 459 for the C18:1, M = 487 forthe C20:1, M = 489 for the di-hydro C20:0 and M = 543 for theC24:1 derivatives of the sphingosine bases. Additional informationwas obtained by chemical ionization and detection of the positiveions with major fragments corresponding to [M + 18 - 214]+ ofM = 691, M = 719, M = 721, and M = 775 for the previouscompounds. It should be noted that in G5b, two different C18:1sphingosines and three C20:1 sphingosines of the same mass areobserved (Figure 2), corresponding to a partial degradationduring the methanolysis step (Pons et al., unpublished data). Theelution of the C:24 derivative implied a prolongation of thetemperature up to 260C. These determinations were in perfectagreement with the data obtained by the MALDI-TOF analysis of


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Fig. 2. Identification of FAMEs ([M+ NH4]+) and sphingosine bases ([M 214 + NH4]+) of G5b ganglioside by GC/MS in the chemical ionization mode(Gas chromatography was performed with a temperature gradient of 10C/min from 90C to 240C. Note the presence of two peaks corresponding to the C18:1sphingosine and three peaks corresponding to the C20:1 sphingosine. Derivatives of monosaccharides are eluted in the first part of the chromatogram (not shown).

these compounds. Attempts were also performed using thedetection of negative ions formed during chemical ionization, adetection that increased the sensitivity of detection of heptaflu-robutyrate derivatives but did not allow the detection of FAME.The ions obtained using this detection system for the HFBderivatives of O-methyl glycosides and of sphingosines were either[M] or [M - 20], the loss of mass of 20 corresponding to theloss of HF. (Figure 3)

The RMR of FAMEs and sphingosines calculated from theseexperiments (Table IV) approached the theoretical RMR calcu-lated from the number of carbon and hydrogen atoms of themolecules. It should be stressed that except for the quantity ofglucose, the molar ratios for these two standard gangliosides wereexact with a precision less than 1%.

Monosaccharide composition of glycoproteins. The GLC profilesof the monosaccharides constituents of glycoproteins are shownin Figures 1b, 1c, 2 and 3, and examples of carbohydratecompositions are presented in Table V. Since the HFB derivativeof the major compounds resulting from the glycosylamine of

GlcNAc involved in the N-glycosidic bond (glucosamine; Maeset al., 1999) was also quantified, the calculation of the molarcomposition of N-glycans was performed relative to thesecompounds. A series of glycoasparagines isolated from themicrosomal fraction of the adult rat brain were analyzed, startingfrom 1 g amount of each glycan. The expression of the differentresults relative to the GlcNAc residue involved in the N-glyco-sidic bond allowed obtaining the composition of the differentoligomannosides without ambiguities. But, because of therelative instability of the glucosamine derivative, the samples hadto be analyzed within 48 h after the evaporation of the acylationmixture. As mentioned above, the peak corresponding to thedi-methyl ester HFB of Asp, resulting from the Asn involved inthe N-glycosidic bond of glycoasparagines, could not be determinedquantitatively since this compound was partially lost during theevaporation of the acylation mixture and during the drying stepin the Ross injector. Nevertheless, the presence of this peak wasindicative that the initial compound was a glycoasparagine andnot a glycan attached to a larger peptide.


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Table IV. Composition of gangliosides

Compound Fuc Gal Man Glc GlcNAc GalNac NeuAc FAME SphingGM1 0.000 2.000 0.000 2.200 0.000 1.000 1.000 1.000 1.005G5b 0.000 2.000 0.000 1.324 0.000 1.000 2.002 1.005 1.011

% of total C18:1 C18:1 C20:1 C20:1 C20:1 C20:0 C22:1 C24:1a

GM1 Sphing 0.00 53.38 0.00 0.00 20.60 26.10 0.00 0.00G5b Sphing 7.91 16.18 4.45 21.06 50.40 0.00 0.00 0.00

% of total C16:0 C18:0 C20:0 C22:0 C24:0 OtherGM1 FAME 0.0 90.0 10.0 0.0 0.0 0.0G5b FAME 6.7 72.6 3.5 8.6 8.6 0.0

aThe C24:1 sphingosine was found in a neutral lipid fraction from synaptosomal plasma membranes. The sphingosine and fatty acid composition allowed abetter interpretation of the MALDI-TOF analyses of the compounds because of the superposition of compounds of the same mass but different FAMEs andsphingosine compositions. The nature of the different sphingosines was established through their mass obtained by GC/MS using the three different modes ofanalysis.

Table V. Composition of standard glycoasparagines and reduced oligosaccharides

N-glycans Fuc Gal Man Glc GlcNAc GalNAc NeuAc GN1Man5GlcNAc 0.00 0.00 4.98 0.00 1.00 0.00 0.00 0.00Man5GlcNAc2Asn 0.00 0.00 5.02 0.00 1.01 0.00 0.00 1.00Man6GlcNAc2Asn 0.00 0.00 6.03 0.00 1.01 0.00 0.00 1.00Man7GlcNAc2Asn 0.00 0.00 7.03 0.00 1.01 0.00 0.00 1.00Man8GlcNAc2Asn 0.00 0.00 8.03 0.00 1.01 0.00 0.00 1.00Man6GlcNAc3Asn 0.00 0.00 6.02 0.00 2.02 0.00 0.00 1.00Fuc-di-antennary 1.00 2.01 3.00 0.00 3.02 0.00 2.02 1.00Fuc-tri-antennary 1.00 3.00 3.01 0.00 4.02 0.00 3.01 1.00Glycoprotein*a 0.000 2.996 3.003 0.000 2.996 0.994 3.003 1.000

(2.743)Reduced O-glycans Fuc Gal Man GlcNAc GalNAc NeuAc GalNAc-OHRp N-4** 1.00 1.99 0.00 0.10 1.00 0.00 1.00

(1.73)Rp N-8* 0.00 2.99 0.02 0.05 0.12 2.01 1.00

(2.74)

The determination was performed starting from 1 g of each compound submitted to methanolysis followed by acylation with HFBAA. Note that thedetermination of the molar ration relative to that of the first GlcNAc residue allows differentiating without ambiguity the different oligomannosidicglycoasparagines with a precision less than 1%. The same is true for complex type N-glycans and hybrid type N-glycans. The reduced O-glycans were isolatedfrom the mucins of the eggs from Rana palustris (Maes et al., unpublished observations).aThis glycoprotein contained the following glycans:NeuAc-Gal-GlcNAc-Man

Man-GlcNAc-GlcNAc-Asn-Prot NeuAc-Gal-GalNAc-Ser-ProtNeuAc-Gal-GlcNAc-ManIn compounds labeled * and **, a defect of the response of Gal was observed when the data were calculated based on the RMRmp (values into brackets). Thisdefect was no more observed when the area of the Gal peaks was obtained taking into accounts all Gal peaks and applying the RMR (see Table II). This was dueto the abnormal increase of the first Gal peak (Rt = 20.04) relative to the others in O-glycans.

The method was applied to a series of reduced oligosaccharideswith defined structures liberated by the reductive -eliminationprocedure. The molar ratios obtained for all these compoundsapproached the theoretical value at less than 1%, and sometimesless than 0.1% (Table V). Surprisingly, in analysis of O-glycans(reduced or not) in which the Gal residue is bound to GalNAc orGalNAc-OH, the first furanic isomer of Gal (Rt = 20.04) showeda significant relative increased compared to the other isomers, itsarea being approximately the double of that obtained for standardof galactose or for complex type N-glycans. This suggested thatduring the cleavage of this particular glycosidic bond, theformation of the O-methyl glycoside of this furanic form of Galis favored, a point that remains to be explained.

Analysis of the possible contaminants issued from glycoproteinisolation procedures. Since the methods of glycoprotein purificationcould introduce intrinsic contaminants, a special care was takento this point. Consequently, deoxycholate, sodium dodecylsulfate, Triton X-100, glycine, and Tris were submitted to thecomplete procedure of methanolysis/HFBAA and analyzed byGLC. A single peak corresponding to deoxycholate was eluted atthe end of the temperature gradient. The peaks of the Tris andglycine derivatives were eluted close to the injection peak andwere in large part lost during the evaporation of the acylatingmixture because of their volatility. Triton X-100 is not significantlydegraded under the methanolysis conditions and gave three smallpeaks without interferences with the monosaccharide derivatives.


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Fig. 3. Chromatogram of the monosaccharide composition of delipized synaptosomal plasma membranes isolated from adult rat brain. Due to the small amount ofGalNAc, it was suggested that O-glycans were minor glycans in these membranes. Note the presence of the peaks corresponding to the GlcNAc residue involvedin the N-glycosidic bond (GN1). No traces of glucuronic acid were found in this material, suggesting that the HNK-1 (glucuronic acid 3-sulfate) epitope is notpresent on these purified plasma membranes. Although the membrane fraction was submitted to a drastic delipidation procedure, FAMEs were still detected inhigh amount, but interestingly, the FAME composition was not the same as that of the lipids extract from the same fraction (Figure 1a). This suggested that animportant quantity of proteins or glycoproteins of these membranes are acylated by fatty acids. Note that after several injections, the separation between the Glcand Gal isomers is almost complete.

In contrast, SDS gave peaks corresponding to compounds with10, 12, 14, and 16 carbon atoms, the major peak of the C:12component interfering with the isomer of mannose. Althoughthe relative proportions of the two Man isomers could not bedetermined, the molar ratio of Man could be determined based onthe -Man area (93.05%). Consequently, these compounds usedfor the purification of membrane-bound glycoproteins did notinterfere significantly with the analysis.

The sensitivity of the method theoretically allowed theanalysis of very small amounts of glycoproteins isolated byelectrophoresis. Therefore, it was of interest to examine ifpolyacrylamide could interfere with the monosaccharidedetermination. For this, a small piece of the polyacrylamide gel(made in Tris-glycine buffer containing 0.1% SDS) was sub-mitted to methanolysis followed by acylation. The completemixture gave only the peaks corresponding to SDS. In order totest the possibility of performing a carbohydrate compositiondirectly on acrylamide gels, two glycoproteins (Thy-1 and CD24isolated as concanavalin A-binding glycoproteins in young ratcerebellum) were isolated by SDSPAGE. The gel was stainedwith Coomassie Brilliant Blue R (solvents were of analyticalgrade for this staining), the stained bands were dissected with alancet and transferred to the methanolysis vessel, and the gelswere dehydrated using repetitive washings in distilled methanol(six times during 10 min). The gels were submitted to methanolysisand after discarding the gels, the samples were evaporated undernitrogen then analyzed after acylation. Although the molar ratiosof the different monosaccharides were identical to the valuesobtained for the same glycoproteins recovered from the gel byelectroelution, the yield of the procedure was at least 10 times less

than expected. This was due to the absence of penetration of themethanolysis reagent into the shrunken polyacrylamide gel (CBBstill remained at the center of the gel after 20 h of methanolysis).Therefore, although the molar composition of the monosaccharidescould be obtained directly on a polyacrylamide gel, the poorefficiency of the methanolysis procedure did not allow thedetermination of the carbohydrate content of a glycoprotein.

This difficulty could be easily circumvented by blotting theglycoproteins on PDVF membranes. After extensive washing withwater, the membrane was stained with Ponceau red, and thenwashed again with water. The stained band was cut, and repetitivelywashed with anhydrous methanol in the methanolysis vessel. Aftermethanolysis, the filter was discarded (after washing with methanol)and the evaporated sample was acylated. In these conditions, themethanolysis procedure allows the quantitative liberation of O-methyl glycosides. No interference occurred if the PDVF mem-branes were repetitively washed in methanol. The technique couldnot be extended to nitrocellulose filters because of the completesolubilization of nitrocellulose in methanol and in acetonitrile.

Because the carbohydrate analysis could be performed oncrude glycoprotein preparation or tissue extracts, we alsoexamined the possible contaminations induced by differentcarbohydrate containing compounds. Consequently, RNA, DNA,and a whole mixture of proteoglycans isolated from adult rat brainin the absence of detergent (Normand et al., 1988), weresubmitted to methanolysis in standard conditions followed byderivatization with HFBAA. Only ribose isomers interfered withthe determination of fucose (Table I, Figure 1), but the presenceof ribose in a sample could be easily detected by the inversion ofthe proportions of the two fuco-pyranose peaks. The small amount


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of derivatives of uronic acids liberated from proteoglycans duringmethanolysis (Table I) did not interfere with the separation ofother constituents (Table I). In contrast, a significant amount ofthese compounds was liberated from branched glycans, in such away that the presence of specific peaks was indicative of thepresence of uronic acids in the glycans (Figure 1b). DNAmethanolysis products resulted in a black suspension uponacylation, but again, none of the peaks interfered with thedetermination of the classical glycoprotein monosaccharides.

Sensitivity of the technique and nanoanalysisThe method allowed the quantitative determination of thecarbohydrate composition of glycoproteins and glycolipids at thelevel of the sensitivity of the FID detection, i.e., at 2550 pmol ofeach monosaccharide injected onto the GLC column, for optimalquantitations. In principle, since the totality of the sample can beapplied to the GLC column, analyses could performed on samplescontaining such amounts of material. However, at this level,dramatic cautions had to be taken with the purity of the reagentsand with the cleaning of the reaction vials, even with new vessels.In our conditions, new vials contained a significant amount ofimpurities that formed volatile derivatives eluted in the area ofFAME, whereas xylose and glucose were detected systematically.Special sequentially redistilled reagents, absence of dust in the

laboratory, and special cleaning of the vessels are needed toperform quantitative determinations starting from picomoles ofeach glycoprotein or glycolipid monosaccharide.

Coupling with mass spectrometry

Although the reproducibility of the GC separation allowed toassign the nature of the compound according to the retentiontimes of the isomers (all isomers of Ara, Rha, Rib, Xyl, Fuc, Gal,Man, Glc, GlcNAc, GalNAc, KDN, Neu5Ac are separated, andseparated from glucitol, mannitol, inositol, N-acetyl-glucosaminitol,N-acetyl-glucosaminitol, and uronic acids; from the fatty acidmethyl esters larger than C14:0; from sphingosine bases; andfrom major contaminants found during glycoprotein and glycolipidisolation), the absolute identification of each compounds insamples with an unknown composition needs mass spectrometry.The HFB derivatives of the O-methyl glycosides of monosac-charides presented a relatively high mass (978 for hexoses, 977for hexosamines, 1275 for sialic acid). Chemical ionization (NH3gas) MS analysis of the compounds performed with detection ofpositive ions, giving the pseudo-molecular ion [M + NH4]+, wasa very secure identification of the families of compounds(Table VI), provided that the mass limit of the apparatus was2000 amu. Unfortunately, most of the routine GC/MS systems areout of this range.

Table VI. Reporter fragments of monosaccharides in the EI/MS and CI/MS modes

Derivatives of EI (70 eV) Specific ions Positive CI(NH3) calc. pseudo Negative CI(NH3)calc. molec. ion observed molec. ion (M+NH4)+ and observed ions observed ions

HFB 69119169Pentoses 752 479265325 770 770 752Deoxy-hexoses 766 279492 784 784 766Hexoses 978 551519337277 996 996 978958Hexosamines 977 276472488702 995 995 977Sialic acid 1275 542602789 1293 1293 n.d.KDN 1276 575515355 1294 1294 n.d.Uronic acids 810 323537597 828 828 810790meso-inositol 1356 715 1374 1374 n.d.mannitol 1358 503453 1376 1376 n.d.GlcNAc-OH 1357 488 1375 1375 n.d.GalNac-OH 1357 488 1375 1375 n.d.GlcN* 963 476348290 981 981963 963943Asp 357 236299357 375 375 357Lys 552 520280 570 570 552Sphingosines C18:1 887 459 905 905691* 887

C20:1 915 487 933 933719* 915C22:1 943 515 961 n.d.C24:1 971 543 989 n.d.C18:0 889 n.d. n.d. 889C20:0s 705 n.d. n.d. 685C22:0s 733 n.d. n.d. 713

For electron impact (EI/MS), the indicated ions correspond to those that allowed the identification of families of compounds or individual compounds. Thescheme of ionization involved primarily a loss of one or two heptafluorobutyric acid(s) (mass decay = 214), and frequently 31 (O-CH3). For uronic acid, anadditional loss of the carboxyl group in the C(6) position occurred. When several ions are reported, the higher mass entity was the more discriminative, butbecause it was present in relatively low proportion, it did not allow the detection of low amounts. Sphingosines gave preferentially ions corresponding to[M 2 214] (M two heptafluorobutyric acids). Furanic forms of oses and uronic acids can be specifically identified by a reporter ion at 525, whereas thefuranic forms of hexosamines are characterized by the ion at 524.In the CI/MS positive mode, the indicated mass corresponded to [M +18]+. * Intense ions were observed corresponding to [M + 18 - 214] for sphingosines. Afragment at M = 867 allows the identification of the derivatives of NeuAc using the Finnigan Automass II mass spectrometer with the mass limit at 1000 amu.In the CI negative mode, the ions corresponded generally to [M]. A loss of mass of 20 was also observed for some compounds corresponding to the loss ofone HF molecule. An ion at M = 809 allowed the characterization of the NeuAc derivative on the Finnigan Automass II device. Sphingosines gave [M] or thesame with a loss of 20. C20:0s and C22:0s were di-hydro-sphingosines found in a tri-sialo-gangliosides isolated from synaptosomal plasma membranes(unpublished observations) in which the hydroxyl group is replaced by a hydrogen atom. n.d., Not determined.


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Fig. 4. Reporter ion analysis in the EI mode of a mixture of pentoses, deoxy-hexoses, hexoses, uronic acids, N-acetyl-hexosamines, and sialic acid submitted tomethanolysis then acylation with HFBAA. Note that specific ions allowed identifying the different families of compounds.

In contrast with the TMS derivatives, HFB derivatives ofO-methyl glycosides of sugars gave secure information under theelectron impact mode, allowing the identification of fragmentsspecific for families of compounds with a routine apparatus witha mass limit of 1000 amu. Because of the presence ofcharacteristic peaks due to the HFB part (M = 69 (CF3+), 119(CF2-CF3+), and 169 (CF2-CF2-CF3+)), it could be ascertainedthat the compound was an heptafluorobutyrate derivative andcontained hydroxyl or amino groups. Furthermore, using theEI/MS mode, specific ions were reproducibly obtained for eachfamily of compounds (Table VI), independent on the quantity ofmaterial. At the present status, the nature of pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid andKDN, aspartic acid, the internal standard lysine, fatty acid methylesters, and sphingosines can be identified using a selectiveresearch of characteristic ions by chromatogram reconstitution.(Figures 2, 4). The presence of specific ions for the furanic formsof the O-methyl glycosides of Fuc, Gal, GalNAc, and uronic acids(legend of Table VI) reinforced the identification of the differentcompounds.

Because of the presence of a large number of fluorine atomsin each derivative, we tested the chemical ionization mode (NH3gas) with a detection of negative ions using the Automass II device(mass limit 1000 amu). The different compounds can be detected byintense ions corresponding to [M] ion and/or [M 20] ion, thelatter issued from an association resonance capture of an electron

with the elimination of a HF molecule. Although this detectiondid not allow obtaining signals for FAME, it appeared to be anextremely sensitive detection technique (more than 20 times thatof the positive ion detection), not only for the HFB derivatives ofO-methyl glycosides, but also for those of sphingosines and ofhydroxylated fatty acid methyl esters.

Conclusions and perspectivesThe method described here for the determination of the mono-saccharide composition of glycoprotein and glycolipids providesimportant advantages compared to those previously described.The major features are simplicity of the technique, the stability ofthe derivatives, and the quasi-absence of interferences byclassical contaminants. The quantitation of the hexosamine andsialic acid content is by far more reliable than that measured bythe classical TMS method, since there is no requirement forre-acetylation. Although the molar response of the HFB derivativesis 50% lower than that of the TMS derivatives, the stability of theHFB derivatives allows one to inject (if necessary), afterconcentrating under a stream of nitrogen, the whole sample on thecolumn. The quantitation available for the GlcNAc residueinvolved in the N-glycosidic bond as a separated compoundprovides an essential information on the structure of N-glycans.As demonstrated elsewhere (Maes et al., 1999), the N-glycosidicbond is cleaved with 96% yield under our methanolysisconditions. Several compounds are produced corresponding


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essentially to the and isomers of glucosamine that are clearlyseparated from the O-methyl glycosides of glucosamine, which arederived from O-linked GlcNAc. Although these compounds can bedetected on chromatograms of TMS derivatives, their quantitationremains impossible because of uncontrolled degradation withinthe GC injector and interference with -Glc; Maes et al., 1999).Because of the absence of large interferences stemming frombuffers and polyacrylamide, the method allows the quantitativedetermination of the glycoprotein monosaccharide compositionwith high sensitivity. Indeed, less than 1 g of glycoproteinpurified from acrylamide or on PDVF membranes is required. Asobserved recently in our laboratory, the exact carbohydratecomposition can be obtained directly from a glycoproteindissolved in phosphate-buffered saline without interference. Forglycoproteins and glycolipids, not only is the carbohydratecomposition accessible but one can also obtain that of the fattyacids methyl esters and that of sphingosines. Specific ions in theEI/MS mode and the molecular mass in the CI/MS modes can bereproducibly obtained, thus nailing down the nature of themonosaccharides, and that of other constituents of glycoconju-gates such as FAMEs and sphingosine bases. Works in the fieldof mass spectrometry are now in progress to identify specific ionsallowing the identification of each isomer of the heptafluoro-butyrate derivatives of all O-methyl glycosides. Because of thereproducibility of the spectra, independent on the quantity ofmaterial loaded of the GC/MS device, it is expected that computerstorage of the data of these spectra can allow a clear and rapididentification of the different compounds using routine GC/MSsystems.

Materials and methods

Chemicals

Standard monosaccharides and sodium deoxycholate were fromSigma Chemical Co. (St. Louis, MO); heptafluorobutyric andpentafluoropropionic anhydrides were from Fluka (Buchs,Switzerland). SE-30 liquid phase and the Gas Chrom Q solidphase were purchased from Applied Science Inc. (State College,PA). The capillary columns (25 m 25QC3/BP1, 0.5 m filmphase) were from SGE France SARL (Villeneuve St. Georges,France). Purified calcinated calcium chloride was from Prolabo(Paris, France) and HPLC grade acetonitrile, TRIS, and acrylamidewere from SDS (Peypin, France). Gangliosides GM1 waspurified from the rat cerebellum (Zanetta et al., 1980), andganglioside G5b was a generous gift of Prof. G. Tettamanti.Reduced oligosaccharides were kindly provided by Dr. E. Maes(Maes et al., unpublished observations). Fatty acid methyl esterswere prepared from the synaptosomal plasma membranes ofadult rat brain (Breckenridge et al., 1972). For use as an internalstandard, lysine hydrochloride (Sigma) was recrystallized from a1 mM HCl solution in water.

Methanolysis

Samples containing the internal standard (0.22 g lysine) werelyophilized in conical heavy walled Pyrex tubes (2.0 ml) withTeflon-lined screw caps. 0.250.5 ml of the methanolysis reagentwas added, and the closed vessels were left for 20 h at 80C. Themethanolysis reagent was obtained by dissolving anhydrousgaseous HCl (up to 0.5 M) at -50C in anhydrous methanolpreviously redistilled on magnesium turnings (Zanetta et al.,

1972). Gaseous HCl was prepared by the dropwise addition ofconcentrated sulfuric acid on crystallized sodium chloride.

AcylationAfter methanolysis, samples were evaporated to dryness under alight stream of nitrogen in a ventilated hood, followed by theaddition of 200 l acetonitrile and 25 l of heptafluorobutyricanhydride (HFBAA) with a pipette with plastic tips. The closedvessels were heated for 30 min at 100C (or better for 5 min at150C in order to have a reflux reaction that derivatizes the tracesof compounds on the vessel wall) in a sand bath. After cooling atroom temperature, the samples can be stored for months at roomtemperature in the closed vessel without need of reacylation.When analysis had to be performed, the samples were evaporatedin a light stream of nitrogen in a ventilated hood in order toeliminate the excess of reagent and the heptafluorobutyric acid(HFBA) formed during the acylation, then taken up in theappropriate volume of acetonitrile; this acetonitrile was stored ina closed vessel in the presence of calcinated calcium chloride inorder to eliminate traces of water. An aliquot of the acetonitrilesolution of the heptafluorobutyrate derivatives (HFB) wasintroduced in the Ross injector of the GC apparatus. Although wedid not observed changes in the RMR of the different derivativesafter storage for 2 days at room temperature of the samples inanhydrous acetonitrile, we do not recommend storing thederivatives in acetonitrile, but in the acylation mixture.

Gas chromatographyFor preliminary studies of the volatility and stability of the HFBFderivatives, analyses were performed on a 3 m long column packedwith 3% SE-30 (Applied Science Inc., State College, PA) on Gaschrom Q (Applied Science Inc.). Injector and detector temperaturewas 260C; and the temperature program was 2C/min from 100to 240C at a helium flow rate of 20 ml/min. For analyticalpurposes, analyses were performed on a Shimadzu GC-14A gaschromatograph equipped with a Ross injector and a 25 m longcapillary column (25QC3/BP1; 0.5 m film phase; SGE FranceSARL; Villeneuve St. Georges (France). Injector and flameionization detector temperatures were 260C and the temperatureprogram was 1.2C/min between 100 and 140C, followed by4C/min from 140C to 240C then maintaining this temperaturefor 10 min. The carrier gas (helium) pressure was 0.8 bar. Thesecond part of the temperature program is only partially involved inthe separation of monosaccharide derivatives (except KDN andsialic acid), but can reveal the presence of additional compounds,especially fatty acids or sphingosines present in glycolipids and alsoin glycoproteins (GPI anchors, myristylation, etc.).

Mass spectrometry

For GC/MS analysis, the GLC separation was performed on aCarlo Erba GC 8000 gas chromatograph equipped with a 25 m 0.32 mm CP-Sil5 CB Low bleed/MS capillary column, 0.25 mfilm phase (Chrompack France, Les Ullis, France). The temperatureof the Ross injector was 260C and the samples were analyzedusing the following temperature program: 90C for 3 min then5C/min until 240C. The column was coupled to a FinniganAutomass II mass spectrometer or, for mass larger than 1000, toa Riber 1010H mass spectrometer (mass detection limit 2000).The analyses were performed either in the electron impact mode(ionization energy 70 eV; source temperature 150C) or in thechemical ionization mode in the presence of ammonia (ionizationenergy 150 eV, source temperature of 100C). The detection was


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performed for positive ions or for negative ions, in separatedexperiments, the latter allowing the quite specific detection ofheptafluorobutyrate derivatives with a higher sensitivity.

AcknowledgmentsWe thank Drs. J.-C.Michalski, G.Strecker, J.Lemoine, E.Maes,O.Kol, and Mrs. A.Copin for providing oligosaccharides andglycoproteins of known monosaccharide compositions and Mrs.C.Alonso for help in iconography.

AbbreviationsAra, arabinose; Rha, rhamnose; Xyl, xylose; Fuc, fucose; Rib,ribose; Gal, galactose; Man, mannose; GlcNAc, gluco-samine; GalNac, N-acetyl-galactosamine; ManNAc, N-acetyl-mannosamine; GalNAc-OH, reduced GalNAc; GlcNAc-OH,reduced GlcNAc; GlcA, glucuronic acid; GalA, galacturonic acid;NeuAc, N-acetyl neuraminic acid; KDN, 3-deoxy-D-glycero-D-galacto-nonulosonic acid; Meso, meso-inositol; Manni, mannitol;Asp, aspartic acid; Lys, lysine. HFB, heptafluorobutyrate; HFBAA,heptafluorobutyric anhydride; TFA, trifluoroacetate; EI, electronimpact; CI, chemical ionization; MS, mass spectrometry.

ReferencesBreckenridge,W.C., Gombos,G. and Morgan,I.G. (1972) The lipid composition of

adult rat brain synaptosomal plasma membranes. Biochim. Biophys. Acta,266, 695707.

Chaplin,M.F. (1994) Monosaccharides. In Chaplin,M.F. and Kennedy,J.F. (eds.),Carbohydrate Analysis. A Practical Approach. Oxford Universtity Press,Oxford, pp. 141.

Chiba,A., Matsumura,K., Yamada,H., Inazu,T., Shimizu,T., Kunusoki,S.,Kanazawa,I., Kobata,A. and Endo,T. (1997) Structures of sialylated O-linkedoligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of anovel O-mannosyl-type oligosaccharide in the binding of alpha dystroglycanwith laminin. J. Biol. Chem., 272, 21562162.

Maes,A., Strecker,G., Timmerman,P., Leroy,Y. and Zanetta,J.-P. (1999) Quantitativecleavage of the N-glycosidic bond in the normal conditions of methanolysisused for the analysis of glycoprotein monosaccharides. Anal. Biochem., in press.

Normand,G., Kuchler,S., Meyer,A., Vincendon,G. and Zanetta,J.-P. (1988)Isolation and immunohistochemical localization of a chondroitin sulfateproteoglycan from adult rat brain. J. Neurochem., 51, 665676.

Zanetta,J.-P. and Vincendon,G. (1973) Gas-liquid chromatography of theN(O)-heptafluorobutyrates of the isoamyl esters of amino acids. I. Separationand quantitative determination of the constituent amino acids of proteins.J. Chromatogr., 76, 9199.

Zanetta,J.-P., Breckenridge,W.C. and Vincendon,G. (1972) Analysis of mono-saccharides by gas-liquid chromatography of the O-methyl glycosides as theirtrifluoroacetate derivatives Application to glycoproteins and glycolipids. J.Chromatogr., 69, 291304.

Zanetta,J.-P., Vitiello,F. and Vincendon,G. (1980) Gangliosides from rat cerebellum:demonstration of a considerable heterogeneity using a new solvent forthin-layer chromatography. Lipids, 15, 10551061.

CGL Heptafluorobutyrate Derivatives Glycosides

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