ISOLATION OF OLIGOSACCHARIDES

Abstract: A method of separating O-linked and/or N-linked oligosaccharide groups from a medium comprising glycoprotein, proteoglycan or glycopeptide to which said groups are linked, wherein said groups are released from said glycoprotein proteoglycan or glycopeptide with the medium and subsequently, in their alditol form, caused to contact a support bearing a reactive dihydroxyboryl group to selectively bind said alditol oligosaccharide groups in the absence of binding other groups to said support and thereafter releasing the bound groups from the support preferably a phenylboronic acid (PBA) column is used for the selective binding and release, and the oligosaccharide fragments coupled after selective oxidation C4-C5 cleavage to lipid molecules to assist detection, identification and other analytical procedures involving chromatographic separation and mass spectrographic techniques.

Description: Isolation of oligosaccharides. This invention is concerned with the characterisation of oligosaccharide groups of glycoproteins, proteoglycans, glycopeptides and the like and has potential application in, inter alia, assessment of glycoproteins for recombinant DNA products. The majority of proteins are post-translationally modified by glycosylation. The degree of glycosylation and the structural pattern of the oligosaccharide chains is dependent on the origin and status of the cell. For example differences in glycosylation pattern are noted for recombinant DNA products grown in yeast or mammalian cell lines, for glycoproteins of normal and diseased tissues and for cells in different stages of differentiation. In addition to having effects on protein stability, conformation and serum half life, the oligosaccharide chains of glycoproteins have specific sequences recognized by antibodies and mammalian cell lectins. These sequences can be present on both ' 0- and N-linked cores. A short form of 0-linked chain also exists (mono - to tetrasaccharide) which may direct an antigenic response to the surrounding amino acid sequence or be part of an oligosaccharide-protein combined antigenic epitope. At present there are few specific isolation procedures for carbohydrates. Possible N-glycosylation of asparagine residues is identified by the presence of the amino acid sequence Asn-X-Ser/Thr. No such consensus sequence is apparent for O-glycosylation of, for example Ser or Thr, and therefore the presence of 0-linked chains is not evaluated. Other glycosylation profiling techniques involving chromatography of the released chains are plagued by radioactive contaminants. In either case a rapid and sensitive method is required to positively detect glycosylation. The microscale structural profiling of N-linked chains of glycoproteins using hydrazinolysis, Biogel P4 chromatography and specific enzyme digestion is well documented. However, methods for microscale sequencing of 0-linked chains of glycoproteins have not been developed. These chains are inherently difficult to purify and analyse in that several types of branching can occur at the protein-linked monosaccharide, e.g. at the core region N- acetylgalactosamine different core region sequences are present and the relatively large chains (found e.g. in mucins) are very heterogeneous. The use of bonded silica sorbent extraction columns has been widely advocated for sample clean-up and concentration for HPLC and has been found to be useful for purification of samples prior to mass spectrometric (MS) analysis. The essence of these preparative techniques is selective binding of the compound of interest. It has been surprisingly found that microscale quantities (e.g.> .g or less) of glycoprotein can be characterised for 0- and/or N-linked glycosylation by sequential chemical release at the corresponding protein backbone linkages and subsequent isolation by specific binding of oligosaccharide alditols to a phenylboronic acid (PBA) column followed by selective elution therefrom. The released oligosaccharide groups may be coupled with a lipid molecule post separation from glycoprotein on the PBA column and, more preferably, a glyceride or phosphatide which possesses at least two hydrophobic polyalkylene chains. The combination of the oligosaccharide and the lipid is referred to as a "neoglycolipid". A neoglycolipid may comprise a long chain fatty acid moiety of from 5 to 50, preferably 5 to 30 and more preferably 10 to 25, carbon atom length, an example being stearyla ine. Simple amino hydrocarbons may also be employed. The amino group is necessary for chemical bonding to the oligosaccharide to form the conjugate. Neoglycolipids may be formed by reacting an oligosaccharide having at least one aldehyde group with an amine having one or more hydrophobic groups. Neoglycolipids of particular interest, have the general formula: Λ R'- NH- CH,-CH,- 0 - P-0 - CH,-CH CH OH 0 0 I I where the integers n, and m have a value w t in tne range 5 to 50, preferably 5 to 30, and desirably 10 to 25. The carbohydrate group R 1 may be derived from a monosaccharide, oligosaccharide, or even a polysaccharide -1 which contains an aldehyde group. A carbohydrate group R ~ which * does not in itself contain the necessary aldehyde group, may be subjected to oxidation, for example using periodate oxidation. Of particular biochemical interest are carbohydrate groups which can be isolated from glycoproteins and proteoglycans by a reduction treatment resulting in a reduced oligosaccharide which contains a methylol group, in which case, to form the necessary aldehyde they may be subjectedTπe a periodate oxidation treatment, most preferred lipids may be derived from phosphatidyl ethanolamine dipalmitoyl 9PPEADP) and its analogues, L-alpha-phosphatidyl-L-serine, L-alpha-phosphatidyl- ethanolamine (PPEA) dilauroyl, PPEA dimyristoyl, PPEA distearoyl, PPEA beta-oleoyl-gamma-palmitoyl. We have now found that a phenyl boronic acid (PBA) column selectively binds reduced oligosaccharides, i.e. those having a sterically unhindered planar array of vicinal hydroxyl groups, separating them completely from glycopeptides, unreduced oligosaccharides and non- carbohydrate material. One particular application area is the purification of oligosaccharide derivatives formed by reductive amination. Reduction amination has been used for the preparation of derivatives for sensitive detection on HPLC (Hase et al., 1984) for increased ionization on MS analysis (Wang et al., 1984) and for antigenic analysis as neoglycolipids (Tang et al., 1986; Tang and Feizi, 1987). The PBA column also has potential applications in other fields where reduced oligosaccharides are obtained from glycoproteins (e.g. by treatment with mild base/borohydride degradation or by hydrazinolysis and reduction, where a simple reduction step can be carried out (e.g. analysis of galatose levels in plasma; Henderson and Fales, 1980) or where reduced oligosaccharides are used as in vivo tracers (e.g. mannitol; Laker et al., 1982). Conditions have been found for the separation of oligosaccharides from their alditols, the separation of alditols from amino acids and glycopeptides and the purification of an oligosaccharides-lipid conjugate (neoglycolipid) formed by reductive amination with phosphatidylethanolamine dipalmitoyl. Phenylboronic acid is an example, when bound to an inert matrix, of an immobilised support comprising the reactive dihydroxyboryl group, which we have found can exclusively bind the planar vicinal diol groups of oligosaccharides in reduced alditol form leading to an effective separation of such from proteinaceous material. The support can be in the form of a silica-bound chromatographic column. The present invention therefore provides (1) a method for the sequential isolation and preferably identification of 0-linked and/or N-linked oligosaccharide chains released from a glycoprotein or peptide sample, and further provides (2) an analytical method for determining oligosaccharide structure in such a sample, (3) a kit-of- parts suitable for carrying into effect method (1) and/or (2), and (4) apparatus comprising means capable of carrying into effect one or more of said methods. According to a first aspect of this invention there is provided a method of separating specific oligosaccharide chains from a glycoprotein or peptide sample containing 0- and/or N-linked oligosaccharide chains which comprises:- (a) treating the sample with a reducing agent in the presence of mild alkali to release 0- linked chains from the sample (b) passing the treated sample through a phenylboronic acid (PBA) column such that 0- linked chains are adsorbed thereon, optionally retaining the effluent for further treatment, and (c) eluting the column with an acidic medium to collect the 0-linked chains as alditols, and optionally (d) subjecting the effluent obtained in step (b) to a process of hydrazinolysis to release N-linked chains, followed by reduction (e) passing the treated sample from step (d) through the same or different phenylboronic acid (PBA) column such that N-linked chains are adsorbed thereon, and (f) eluting that column with an acidic medium to collect the N-linked chains as non-N-acetylated alditols. It is preferred that a fluorescent or radioactive label is introduced on β-elimination or on reduction. Preferred agents include, for example, a tritium-labelled borohydride or fluorescent cyanoacetamide. Preferably, the sample is applied to the support, e.g. the PBA column, in alkaline pH and the support is subsequently eluted under acidic pH. In a second aspect the invention also provides an analytical method for determining oligosaccharide structure in a glycoprotein or peptide sample containing 0- and/or N- linked oligosaccharide chains which comprises a separation method as defined in the first aspect (preferably including the use of labelled reducing agents in steps (a) and/or d) followed by:- (g) treating the eluate obtained in step (c) above with a mild oxidising agent such as periodate to cleave the terminal (protein- proximal) monosaccharide chain and form the corresponding aldehydes, (h) treating the aldehyde by reductive amination to form lipid or amine containing molecules (i) subjecting the product, formed in step (h) to an analytical technique capable of separating and identifying characteristic molecular structures of 0- and/or N-linked oligosaccharide derivatives, which technique may be performed by comparison with previously chromatographed standards of such derivatives. In a third aspect the invention provides an analytical kit for carrying out the methods of the first and/or second aspects of the invention which comprises:- (j) a phenylboronic acid column, such as a commercially available PBA mini-column, (k) chromatographic analytical apparatus capable of identifying characteristic derivatives or fragments of 0- and/or N-linked oligosaccharides, (1) chromatographic standards identif ing a plurality of such derivatives of fragments, and (m) instructions for use of the kit sufficient to perform methods (1) and (2) above. The specific binding of reduced oligosaccharides by PBA ensures that proteins, reagents, salts and other contaminants do not confuse the diagnosis of N- and/or 0- glycosylation. Specificity is achieved by the stereochemistry of PBA which binds only to vicinal planar hydroxyl groups which are present in oligosaccharide alditols and catecholamines. Detection of the latter group of compounds is excluded because they would not be labelled by the reduction or reductive amination steps (Reaction Scheme 1) . The present invention embraces a method of separating 0-linked and/or N-linked glycosylation and facilitating their subsequent identification by combining the selective oxidative cleavage of the C4-C5 bond of the protein-proximal monosaccharide, together with the high resolving power of t.l.c. The mild oxidising conditions selected are most preferably highly specific, i.e. selective for the alditol moiety, without oxidising other parts of the oligosaccharide groups. This can be achieved by using periodate at relatively low concentration, conducting the oxidation step at low temperature and in the presence of a quenching agent to inactivate any excess oxidant. Such a mild oxidising step is of particular advantage prior to reductive amination in minimising side reactions and unwanted reaction components. The mild periodate oxidation step has surprisingly been found to specifically cleave the C4-C5 bond of the terminal monosaccharide unit e.g. N-acetylgalactosaminitol disubstituted at C3 and C6, or monosubstituted at C3 or C6. In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after mild periodate oxidation and conjugation by reductive amination to the aminolipid phosphatidylethanolamine dipalmitoate (PPEADP), see Figure 2 oligosaccharides branched at the core N-acetylgalactosaminitol each give two derivatives. Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin layer chromatography and can be sensitively analysed by liquid secondary ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the N-acetylgalactosaminitol. Such neoglycolipids therefore confer considerable advantage for biological and immunological assessment. Characterisation of the products formed from a series of partially purified di- to hexasaccharide alditols released from mucin glycoproteins was carried out and confirmed the utility of the method. LSIMS of mucin derived oligosaccharide alditols The mechanism of periodate oxidation of a 3,6- disubstituted galactosaminitol, followed by reductive amination of the resulting aldehydes with PPEADP is shown in the reaction illustrated in the results observed for fractions Rl, 01 and Nl. The di-to hexasaccharides isolated from human meconium glycoprotein analysed are shown in TABLE 1. The fragments expected by their periodate oxidation and reductive amination are also shown in TABLE 2 together with the masses of their [M - H]- ions. As there is only one vicinal diol in the 3,6-disubstituted aminoalditol, oxidative cleavage of the C4-C5 bond was observed. The following description of oligosaccharide profiling is given by way of example only. Radioactively or fluorescently labelled oligosaccharide alditols isolated from the PBA column can be treated by mild periodate oxidation and the resulting aldehydes specifically formed at the "protein linkage" sugar coupled by reductive amination to lipid by a known procedure or any amine containing molecule (e.g. those giving sensitive t.l.c. or .s. detection). As shown in Reaction Scheme 2 for 0-linked chains, this results in two fragments characteristic of branches at the C6 and C3 positions of reduced N-acetylgalactosamine (GalNAc-ol) which can be separated and detected on t.l.c. These techniques can resolve oligosaccharides by composition and linkage differences thus providing a profile not only of oligosaccharide size and branching pattern but also of specific structures. The specific isolation of oligosaccharides prior to the synthesis of neoglycolipids for antigenic and functional studies of oligosaccharides is most desirable. The present method ensures that antigenic or receptor specificity is only assigned to oligosaccharides. All of the oligosaccharides which bind to PBA will be oxidised by periodate which is a necessary step in the production of neoglycolipids. This is because both PBA and periodate require access to planar vicinal hydroxyl groups. The PBA column can potentially be used to isolate reducing mono- and oligosaccharides from body fluids. Only the reducing sugars present in such mixtures would be labelled by reduction or reductive amination and subsequently isolated by binding to a PBA mini-column. PPEADP derivatives give good thin layer chromatographic separations including those of isomers. This should allow future characterisation of products by t.l.c. alone. Because the derivatives are held to the silica surface by hydrophobic forces, in-situ enzyme degradation can be carried out on each branch separately to provide further sequence and immunoreactivity assignments. The major advantage of the technique compared to other structural analysis regimes is that it gives unambiguous sequence information on the C6 and C3 branches of GalNAc-ol. For oligosaccharides previously documented this can be achieved by NMR analysis. For large structures the NMR data is more difficult to analyse as monosaccharides distant from GalNAc-ol can sometimes greatly influence the chemical shifts of this moiety e.g. substituents on Gal linkedj3> 1-3 to GalNAc-ol. Conditions have been achieved for mild periodate oxidation of the oligosaccharide alditols released from protein by β-elimination with concomitant reduction. The resulting aldehydes may be coupled to dipalmitoyl phosphatidylethanolamine by reductive amination by a known procedure and following chromatographic separation on Aluminium backed t.l.c. plates the derivatives can be analysed by direct liquid secondary ion mass spectrometry. This method permits elucidation of the sequence, ^ linkage positions and core region branching patterns of 0-linked chains of glycoproteins on less than 5 nmole oligosaccharide and is therefore a powerful tool for_ their microscale profiling. Studies of a series of oligosaccharide fractions derived from human meconium glycopeptides show the present invention has utility in characterising oligosaccharide mixtures particularly those that are too complex for elucidation by conventional MS and NMR alone. Neoglycolipid derivatives of several oligosaccharides (di- to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols were investigated. Sequence information was obtained leading to the structural assignments of two major hexasaccharides enabling several less abundant components (penta- and hexasaccharides) to be identified. With a fraction that was too complex for analysis of the free alditols by MS and NMR alone, sequences for one linear and three branched pentasaccharides could be proposed. . The present invention introduces a new approach to the microscale structural profiling of 0-lin ed oligosaccharides. In particular the high chromatographic resolution of the neoglycolipid products arising from fission at core N-acetylgalactosaminitol, their excellent ionisation properties and the sensitivity of detection at the silica surface by LSIMS enable unambiguous assignments to be made for sequences within the 3- and 6- linked branches. The previously developed method [4] for mild periodate oxidation of vicinal planar hydroxyl groups in the reduced-end monosaccharide has been applied and extended to the analysis of the individual neoglycolipid products. The oxidative cleavage of the core N-acetylgalactosaminitol at the C4-C5 bond described here accounts for two observations in the earlier study [4 " }: (a) the successful derivatisation of the oligosaccharide alditol Nl in situ after TLC and (b) the preservation of reactivity of the products with the monoclonal antibody, anti-I Ma [4], Of the two lipid derivatives thus obtained: Galβl-4GlcNAcβl 6-OY .3-OX Galβl 7 the former would contain the epitope for this antibody which is known to consist of Galβl-4GlcNAcβl-6-CH 2 - [ 10, 11 ] . Previous studies of oligosaccharide alditol oxidations by mild periodate [12] have indicated that glucitols substituted at C3 or at C4 have differing susceptibilities to fission of C5-C6 and C1-C2 sites. In the present invention, oligosaccharides with reduced end N-acetylgalactosaminitol have been observed to exhibit exclusive fission of the C4-C5 bond of branched oligosaccharides and that the C5-C6 bond which is available in linear oligosaccharides with 3-substitution is not a site of primary oxidation. With the oligosaccharides tested we have found no mass spectrometric evidence for degradation of the oligosaccharides outside the core N- acetylgalactosaminitol. Although there is scope for improved chromatographic separation, the majority of neoglycolipid products arising as a result of core region cleavage were well resolved by the TLC system used. For example, the isomeric pair Galβl-3/4GlcNAcβl-3-0X chromatographed at positions 6 and 7, (TABLE 2) respectively, and the derivative GlcNAcc(l-4Gali31-3-OX (position 4) was well separated from two derivatives at positions 5 and 8 which according to the LSIMS data also had the sequence HexNAc- Hex-3-OX. There were several instances where more than one derivative was present at the same position e.g. positions 8 to 10, 12 and 13 but in most cases individual components could be identified by LSIMS analysis. The sequence data derived by LSIMS analysis of the neoglycolipid derivatives are in excellent agreement with the assignments that could be made previously (Table 1) by MS and NMR analysis of the free oligosaccharide alditols. With several oligosaccharide fractions which could only be partially characterized as the free oligosaccharide alditols the sequence information obtained by TLC-MS of neoglycolipids enabled structural assignments to be made. Thus essential information was obtained for the characterization of the penta- and hexasaccharides (TABLE 1) in fractions K3(4) and K4(3) and of several minor components in fractions K2(3), K3(4), K4(3), K2(3), K6(2) and K6(3). In the case of fraction K3(2), which was too complex for analysis of the free oligosaccharides by MS and NMR alone [3], sequences within one linear and three branched S U BS T I TUTESHEET - 12a - pentasaccharide isomers could be proposed. In order the invention may be more readily appreciated reference is now made to flow charts, reaction schemes, tables and drawings by way of non-limiting examples only, wherein in the tables and other drawings:- FIGURE 1 shows a chromatographic standard for comparison with samples to be evaluated for identification by TLC, FIGURE 2 a mixture of reduced and non-reduced glucose oligomers derived from dextran by hydrolysis fractionated on a PBA column under condition A. The figure shows TLC (solvent 1) with orcinol detection of aliquots of the eluted fractions. G1-G7, oligosaccharides with 1-7 glucose units and the corresponding alditols having a slightlylower ^ compared to the parent reducing oligosaccharides; S, non-reduced dextran hydrolysate standard; SR, reduced dextran hydrolysate standard; 1-8, fraction number. FIGURE 3 a mixture of reduced (GM 1 -ol) and non-reduced octasaccharide GM.-_ fractionated on a PBA column under condition A. The figure, shows TLC (solvent II) with orcinol detection of aliquots of the eluted fractions 1-10. FIGURE 4 the triasaccharide alditol GlcNAcβl-6[Gal l- 3]GalNAc-ol passed through a PBA column under condition A. The figure shows TLC (solvent II) with orcinol detection of aliquots of " the eluted fractions 1-10. ' FIGURE 5 a mixture of 20 common amino acids and reduced dextran hydrolysate on a PBA column under condition B. The figure shows TLC (solvent)) of aliquots of the eluted fractions 1-9. (a) Ninhydrin detection, (b) orcinol detection. FIGURE 6 TLC (solvent II) of aliquots of the eluted fractions (1-9) from a PBA column run under condition B.(a) A mixture of GA, Asn, Gly-Ser and fetuin detected with ninhydrin. (b) A mixture of GM- j _-ol, GM- j _ and NGGA detected with orcinol. FIGURE 7 a mixture of reduced (LacNAc-ol) and unreduced (Lac-NAc) N-acetyllactosmine fractionated on a PBA column under condition B. The figure shows TLC (solvent II) with orcinol detection of aliquots of the eluted fractions 1- 10. FIGURE 8 a mixture of reduced (2-3ol and 2-6ol) and unreduced (2-3 and 2-6) NeuAc<X2-3(6)Gal|31-4Glc fractionated on a PBA column under condition B. The figure shows TLC (solvent II) with orcinol detection of aliquots of the eluted fractions 1-10. FIGURE 9 the reaction mixture obtained in the reductive amination of lactose with PPEADP and soium cyanoborohydride fractionated on a PBA column under condition C. The figure shows TLC (solvent III) with primulin detection of aliquots of the eluted fractions 1-14. FIGURE 10 proposed mechanism of action of the PBA column, (a) Activation of matrix bound PBA to give phenylboronate. (b) Covalent bonding of planar vicinal diol to phenylboronate to give complex. (c) Release of diol from complex by dilute acid. FIGURE 11 TLC of 12 oligosaccharide alditol fractions derived from human meconium glycopeptides. Each lane contained approximately lnmol oligosaccharide. Large arrow indicates origin. Staining was with orcinol. The continuous band (indicated by small arrow) which stained yellow is a solvent artefact. FIGURE 12 TLC of the neoglycolipids derived from the di-, tri-, and tetrasaccharide alditols Rl, 01 and Nl. Each lane contained 1-2 nmol of carbohydrate. Panel A, orcinol staining; panel B, primulin staining; arrow indicates origin; L, excess lipid. The numbered hands at positions 1,2 and 8 were analysed by LSIMS; and FIGURE 13 shows primulin stained chromatograph of the neoglycolipids obtained from the oligosaccharide fractions K5(3), K2(3), K3(4), K6(2), K6(3) , K4(2), K4(3) and K3(2) in panel A, and from standard oligosacchardies designated 03 and 07, (panel B). Each lane contained l-2nmol of oligosaccharide. Bands at positions 1-13 are indicated; arrow indicates origin. - - Flow chart for isolation of 0- and N-linked chains Glycoprotein 1(having both 0- and N-linked chains) Mild alkali/borohydride treatment for release of 0-1inked chains [Carlson degradation; Iyer and Carlson ABB 142(1971) 101-105] (i) 0.05M NaOH in 1M NaBH 4 *, 50 C, 16 hrs (ii) Neutralisation and lyophilisation (iii) Taken up in alkali and applied to a prewashed PBA mini-column. Alkali Acid Fall through Specific eluate Glycopeptides with 0-linked chains as attached N-linked their alditols chains, salts Release of N-linked chains and reduction ( ) Hydrazinolysis Anhydrous hydrazine, 100° C.16 hrs (ii) Evaporation of hydrazine over H2SO4 (iii) 0.05 M NaOH, 0.02M NaBH 4 *, 4°C, 16 hrs (iv) Neutralisation and lyophilisation (v) Taken up in alkali and applied to a prewashed PBA mini-column \ Alkali Acid Fall through Specific Eluate Amino acids N-linked chains as their Salts non-N-acetylated alditols *NaB[°H] 4 for radioactive detection or use of cyanoacetamide for fluorescent detection [Crowther and Wetmore Anal Biochem 163 (1987) 1970-174] - 16 - Reaction Scheme 1 O-linked oligosaccharide chains 3- 6- or 3- and 6- linked GalNAcc --Ser( Thr) PRoiεiN treatment wi Ith mild alkali/borohydride — Specific interaction of vicinal planar ( OH ) OH groups with PBA. Binding also occurs ( OH ) in absence of substituents at C6 or C3 — O (No substituents have yet been found HCNHAc linked at C4 of GalNAcctl-Ser(Thr) I CH 2 OH N-linked oligosaccharide chains N-linked * GlcNAcβl-Asn pβOTElW hydrazinolysis and reduction CH 2 0H in absenc 1e of substituent at C6* CH(OH) interaction with PBA —OCH / CH(OH) (NH)CH CH 2 0H * Fucose residues occurring at C6 have to be removed with et-fucosidase or mild acid hydrolysis after hydrazinolysis, No other subtituents on GlcNAcβl-Asn have been found to date - 17 - Reaction Scheme 2 Glycoprotein (i) Mild alkali/ 3 H sodium borohydride (ii) PBA column separation / Fall through Specific Eluate Peptides, Released 0-linked chains glycopeptides as their labelled alditols -0-CH 2 CH(OH) specific binding CH(OH) to PBA in alkali -O-CH I CHNHAc CH 3 H(OH) mild periodate oxidation CHNHAc CH^HfOH) reductive amination (CH=0 CH 2 R) and t.l.c. See e.g. Figure 1 - 18 - EXAMPLES PART (1) Materials. The PBA Bond Elut columns [Analytichem Int., Harbor City, USA] were obtained from Jones Chromatography [Hengoed, Glamorgan, UK]. Thin layer chromatography plates were 5μm silica [Merck from BDH, Poole, Dorset, UK]. Lactose, N- acetyllactosamine, sialyllactose of human milk, fetuin, porcine submaxillary mucin, phosphatidylethanolamine dipalmitoyl(PPEADP) , the dipeptide Gly-Ser (GS), amino acids and dextran T200 were from Sigma Chemical Co. Ltd [Poole, Dorset, UK] . A mixture of glucose oligomers was obtained from dextran T200 by hydrolysis in 01M H 2 S0 4 for 16h at lOO'C. The octasaccharide Galβl-4GlcNAcβl-2Manc(l. δ Manβl-4GlcNAc(GM 1 ) from the urine of a patient with GM- j _ gangliosidosis and NeuAcc2-3Galβl-4GlcNAcβl-Asn (NGGA) from the urine of a patient with aspartyijlucosaminuria were kindly supplied by Drs S. G. Strecker and J.C. Michalski [U.S.T.L. Flanders Artois, Villeneuve, France]. GlcNAc-Asn (GA) was chemically synthesized according to the method of Spinola and Jeanloz (1970). The trisaccharide GlcNAcβl-6[Galβl-3]GalNAc-ol was obtained from meconium glycopeptides by base-borohydride degradation (Hounsell et al. , 1985). Reduction of oligosaccharides was carried out using 20 mM sodium borohydride for 24 h at 4 * C followed by neutralization with glacial acetic acid, deionization on a column of Dowex 50X12 (H+ form) and evaporation several times from methanol. Base-borohydride degradation of fetuin and porcine submaxillary gland mucin was carried out as described previously (Hounsell et al., 1984). Thin layer chromatography. Glucose oligomers of the dextran hydrolysate and amino acids were chromatographed in solvent system propan-1-ol/water 8:3 v/v (Solvent I). Other oligosaccharides, GA, Gly-Ser and glycopeptides/glycoproteins - 18a - were chromatographed in a solvent system of ethanol/butanol/ pyridine/water/acetic acid 100:10:10:40:3 v/v (Solvent II). Hexose containing material was detected by spraying with orcinol reagent (2% orcinol w/v in ethanol containing 5% sulphuric acid) and heating at 105"C for 5 min. Material containing amino acids was detected by spraying with ninhydrin reagent (0.3% w/v ninhydrin and 3% v/v acetic acid in butan-1- ol) .and heating at 105 * C for 5 min. PPEADP and PPEADP derivatives (neoglycolipids) were chromatographed in solvent system chloroform/methanol/ water, 60:35:8 v/v (Solvent III) and detected by spraying with primulin reagent (1 mg primulin/100 ml acetone/water, 4:1 v/v) and visualizing under long wave UV. Use of the PBA column. The PBA column was activated by consecutive washing with 2 x 1 mL of methanol, 1 mL of 0.1 M HC1, 2 x lmL water and 4 x 1 mL of 0.2 M NH 4 0H. Samples were then applied to the column under one of three conditions (A-C described below) . Condition A: the column was washed with 2 x 1 mL water and the sample applied in 200 μL water (fraction 1) followed by elution with 3 x 200 μL water (fractions 2-4), 5 x 200 μL 0.1 M acetic acid (fractions 5-9) and 1 mL 0.1 M HC1 (fraction 10). Condition B: the sample was applied in 200 μL 0.2 M NH 4 OH (fraction 1) without a prior water wash of the column and elution carried out with 2 x 100 μL 0.2 M NH 4 0H (fractions 2 and 3), 2 x 200 μL water (fractions 4 and 5) and 4 x 0.1 M acetic acid (fractions 6-10). Condition C: the column was washed with 2 x 1 mL water, 2 x lmL methanol and 2 x lmL chloroform/methanol (1:1 v/v). The products of a reductive amination reaction between lactose and PPEADP (details to be reported elsewhere) where then applied in 350 μL chloroform/methanol (1:1 v/v) (fraction 1) and elution carried out with chloroform/methanol (1:1 v/v) (5 x 200 μL; fractions 2-6), chloroform/methanol/water (30:70:30 v/v) vol (4 x 200 μL, fractions 7-10) and chloroform/methanol/0.1 M acetic - 18b - acid (30:70:30 v/v) (6 x 200 μL; fractions 11-16). Separation of oligosaccharides from alditols : Figure 2 shows that for oligosaccharides of increasing molecular size those having glucose at the reducing end are not retained by the PBA column in mild alkali (eluted in fractions 2 and 3) whereas the corresponding alditols are retained and specifically eluted with decreasing pH (in fractions 6 and 7). The same behaviour (Fig. 3) was shown by the glycoprotein derived octasaccharides GM-^ which could be also separated from its alditol, the latter being retained by the PBA column. A typical oligosaccharide alditol, GlcNAcβl —6[Galβl-3]Ga3NAc-ol, obtained by base-borohydride degradation of mucin-type glycoproteins (Hounsell et al., 1985), was also retained by the column and eluted in fractions 5-9 (Fig. 4). The column therefore is generally applicable to the separation of oligosaccharides from their alditols. Separation of oligosaccharide alditols from amino acids, peptides and glycopeptides. In order to investigate whether reduced oligosaccharides could also be separated from proteinaceous material, first a mixture of the 20 common amino acids was passed down the column under the identical conditions of Figs. 2 to^(condition A). Some retention of the more basic amino acids (Arg, Lys, hydroxyLys and His) was observed but this could be » obviated by applying the samples in 0.2M NH 4 0H (having omitted the water wash after the 0.2 M NH 4 OH column activation step; condition B). When a mixture of the amino acids and the reduced dextran hydrolysate was applied to the PBA column together in 0.2 M NH 4 0H, a complete separation of amino acids and alditols was achieved (Fig. 5). In addition, the glycoprotein fetuin, the glycopeptides GA and NGGA and the dipeptide GS were not retained by the column in alkali (Fig. 6). The PBA column was therefore shown to have potential for the purification of alditols from peptides and glycopeptides. The chromatography under condition B of base-borohydride degradation of fetuin (Hounsell et al., - 18c - 1984) and porcine submaxillary mucin gave hexose positive bands in fractions 1-3 and 8-10, indicating a separation of larger glycopeptides from alditols (results not shown). Under condition B there was some retention of non- reduced oligosaccharides, e.g. GM-^ (Fig. βb) and LacNAc (Fig. 7). Therefore, although all the alditols tested could be separated from non-sugar components under condition B, condition A is required to completely separate some oligosaccharides from their alditols. Condition B was better suited than condition A to separation of sialyllactose from sialyllactitol. Under condition B the non-reduced isomers eluted in fractions 1-3 and the alditols in fractions 3-8, giving greater than 90% purification (Fig. 8, whereas under condition A the respective fractions were 1-2 and 2-3 giving less than 50% purification (results not shown). Complete separation of these has been achieved by increasing the concentration of ammonia in condition B to 0.4 M which gives elution -of the non-reduced isomers in fractions 1-2 and the alditols in fractions 4-5 (results not shown) . - 19 - Careful choice of binding anά elusion will therefore allow tailoring of separations for specific needs. Purification of oligosaccharide derivatives An additionalapplication of the FBA column is the purification of oligosaccharide derivatives formed by reductive animination which results in opening of the reducing end sugar ring to give acylic vicinal hydroxyl groups can bind to the PBA. An example of this purification is given in Fig. 9 for the reaction mixture from reductive amination of lactose with PPEADP. This mixture contained excess PPEADP, derivatized lactose and salts derived from sodiurύ cyano borohydride in chloroform/methanol (1:1). Separation on the PBA column under condition C gave FPΞADP in fractions 1-4 and the lactose-FPEAD? derivative in fractions 6-14). Thus the derivative was completely separated from the other components in the mixture. We have also shown (G. Cash ore, M.S. Stoii and E.F. Hounsell, unpublished results) that the PEA clean-up is equally applicable to the products of reductive amination with less hydrophobic re άiits sucn as s * cβr. nis cierivatizaticn n been proposed by Wan et al. (1934) ϋo give a compound with increased ioni <__."__ion in 1.3 . RE U TS OF EXAMPLES PART (1) Our results indicate that the PBA column specifically binds to copianar vicinal hydroxyl groups. Conditions have been found where binding of the acyclic d ' løls present in the reduced carbohydates and lactose reductively coupled to PPEADP occurs in the absence of binding of the non-coplanar diol groups in pyranose rings. Further, the sterically hindered glycerol side chain of sialic acid linked Q2-3 or QL2-6 to galactose was not bound by the column thus allowing for the separation of sialylated alditols from non-reduced sialylated - 19a - oligosaccharides, non-carbohydrate material and glycopeptides. A less specific and weaker mechanism of binding to PBA appears to be by cation exchange which would explain the observed binding of strongly basic amino acids, i.e. those containing basic centres other than the zwitterionic amino group. This binding was abolished by raising the pH to reduce the ionization of the basic groups which in addition increased retnetion of sialylated alditols and gave some retention of non-reduced neutral oligosaccharides. The order of retention on the column was therefore neutral alditols > strongly basic amino acids> weakly basic amino acids > sialylated alditols > neutral or sialylated unreduced oligosaccharides. The mechanism of binding shown in Fig. 10 is similar to that reported for the isolation of catecholamines (Smedes et al. , 1982). High pH favours stronger binding. By suitable choice of pH the required separations can be achieved. REFERENCES FOR EXAMPLES PART " (I) Hase, S., Ibuki, T. and ik≤naka, T. (1984). J. Biochem: 95, 197. Henderson, J.M. and Fales, F.W. (1980). Clin. Che . 26, 282. Hounsell, Ξ.F., Pickering, N.J., Ξtoll, M.S., Lawsoπ, A.M. and Fei∑i, T. (19S4). Biochem. Soc. Trans. 12, 607. Hounsell, E.F., Lawson, A.M., Feeney, J. , Goci, H.C., Pickering, N. J., Stoll, M.S., Lui, S.C. and Feizi, T. (1985). Eur. J. Biochem. 148, 367. Laker, il.F., Bull, K.J. and Menzies, I.S. (1SS2). J. Ciin. Invest. 12, 435. Smedes, F., Kraak, J.C. and Foppe, H. (1982). J. Chromatogr. 231, 25. Spi ola M. and Jeanloz, R.W. (197C). J. Biol. Chem. 245, 4158. Tang. P.W., Gooi, H.C., Hardy, M. , Lee, Y.C. and Feizi, 1. (l ζlή - 19b - Biochem. Biophys. Res. Commun. 132, 474. Tang. P.W. and Feizi, T. (1987). Carbohydrate Res, 161, 133. Wang, W.T., LeDonne, N.C., Ackerman, B. and Sweeley, CC. (1984). Anal. Biochem. 141, 366. - 20 - EXAMPLES PART (II) Materials: HP-TLC plates (aluminium-backed 5μm silica, Merck) were from BDH, Poole, Dorset, UK. C 18 Bond Elut columns (lOOmg in 1ml) were from Jones Chromatography, Hengoed, Glamorgan, UK. Sodium cyanoborohydride, PPEADP, primulin, orcinol, lactose and imidazole were from Sigma Chem. Co. Ltd., Poole, Dorset, UK. Chloroform, ethanol, methanol, sodium periodate, butan-2,3-diol, nitrobenzyl alcohol, tetramethylurea and triethanolamine were Analytical Reagent grade. Imidazole buffer was 40mM adjusted to pH 6.5 with hydrochloric acid. Primulin reagent was 0.001% in acetone/water, 4:1 by vol. Orcinol reagent was 0.2%. (wt/vol) in ethanol containing water (5.6%, by vol) and concentrated sulphuric acid (11.1%, by vol). The oligosaccharides investigated were di- to hexasaccharide alditols released from human meconium glycopeptides and fractionated by Bio-Gel P4 chromatography and HPLC. These were designated Rl, 01, Nl, K2(3), K2(4), K3(4), K4(2), K4(3), K5(3), K6(2) and K6(3). The structures previously identified in each fraction [3,6,7] are shown in Table 1. An additional fraction, K3(2), which could not be characterised fully by classical methods due to its heterogeneity was also investigated in the present study. TLC of the twelve oligosaccharide fractions in the solvent system butan-1- ol/acetone/water (6:5:4, by vol) is shown in Fig 11 Standard oligosaccharides obtained by chemical synthesis Galβl-3GlcNAcβl-3Gal and Galβl-4GlcNAcβl-3Gal (designated 03 and 07, respectively) were donated. Periodate oxidation of the oligosaccharide alditols: The alditols (5-13nmol) in water were evaporated to dryness under nitrogen and oxidised in the dark with sodium periodate (5μg; 23nmol) in imidazole buffer (4μl), at 0 * for 5 min. Excess oxidant was reacted with a quenching agent which was present viz butan-2,3-diol (4.4 g; 48nmol) in water (0.3μl) under the same conditions for a further 40 mins. - 21 - Conjugation of oligosaccharides to PPEADP by reductive amination: To the reaction mixture from periodate oxidation (4.3μ\.) was added PPEADP (78μg; 113nmol) in chloroform/ethanol (1:1, by vol; 78μl). The mixture was heated at 50 * for 2h. Sodium cyanoborohydride (40μg; 635 nmol) in methanol (4μl) was added and the mixture heated at 50 * for a further 16h. The solvent was then evaporated under a stream of nitrogen. The reference oligosaccharides 03 and 07 were conjugated to PPEADP as described previously [8]. Isolation of lipids on a C-^g Bond Elut column and TLC: The column was washed successively with methanol (1ml), chloroform/methanol/water (60:35:8, by vol; 1ml) and chloroform/methanol/water (15:70:30, by vol; 2ml). The residue after evaporation of the reaction mixture from reductive amination was redissolved in chloroform/methanol/water (15:70:30, by vol; 400μl) and applied to the column which was then washed with the same solvent (2 x 400μl). The lipids (neoglycolipids and excess PPEADP) were eluted with chloroform/methanol/water (60:35:8, by vol; 2 x 1ml). The residue of this eluate after evaporation was redissolved in the same solvent to give a carbohydrate concentration of approximately O.lnmol/μl. For TLC 1-2 nmol of the carbohydrate-PPEADP conjugates was applied as a 5mm band and developed in solvent system chloroform/methanol/water (130:5:9, by vol). The lipids were visualised by spraying with primulin reagent and viewing under long-wave UV and carbohydrate (hexose) was visualised by spraying with orcinol reagent and heating at 105 * for 5 min. Liquid Secondary Ion Mass Spectrometry: After TLC the neoglycolipid bands were cut out as strips (5x1.5mm) on the aluminium backed silica gel and placed on a standard stainless steel LSIMS target probe. A matrix of tetramethylurea/triethanolamine/meta-nitrobenzyl alcohol (2:2:1, by vol; 2μl), and chloroform/methanol/water (25:25:8, by vol; - 22 - 2μl) was placed on the silica gel strip and the sample ionised in the negative ion mode using a caesium ion gun (25keV, 0.5μA emmission) on a VG Analytical ZAB2-E mass spectrometer operating at 8keV accelerating voltage and 2000 resolving power. Mass scans were made at 30 sec/decade in the multichannel acquisition mode. The characteristics of the mass spectra of oligosaccharides linked to PPEADP have been discussed previously £9]. - 23 - RESULTS OF EXAMPLES PART (II) The tetrasaccharide alditol fraction Nl had previously been conjugated to PPEADP after TLC followed by in situ oxidation with periodate [4]. LSIMS analysis was undertaken for the neoglycolipid products obtained from fraction Nl as well as those from the di- and trisaccharide fractions Rl and 01 after oxidation and conjugation in solution followed by TLC. Thereaf er detailedTLC-MS analyses were performed to explore the degree of structural information obtainable with the several partially purified penta- and hexasaccharide alditol fractions derived from human meconium glycopeptides. Fractions Rl, 01 and Nl: The neoglycolipids derived from oligosaccharide fractions Rl, 01 and Nl are shown in Fig. 12. Oligosaccharide Rl gave a single neoglycolipid band at position 1 stained by orcinol and primulin, indicating the presence of both hexose and lipid moieties. The results of negative ion LSIMS of this band (not shown) were consistent with the following reaction scheme in which cleavage occurs across the C4-C5 bond of GalNAcol. R 2 -3-OX CH 2 OCO(CH 2 ) 14 CH 3 R, : 6-linked saccharide or H Lipid : CHOCO(CH 2 ) 14 CH 3 R : 3-linked saccharide or H CH 2 OP(0)(OH)OCH 2 CH 2 NH- - 24 - Thus, a molecular ion [M-H] m/z 997.6 and a fragment ion m/z 835 arising from loss of Gal with hydrogen rearrangement were observed consistent with the PPEADP derivative of the Cl to C4 fragment of GalNAcol with galactose linked at C3 (Galβl-3-OX, Table 2). The fragment of GalNAcol arising from the C5 to C6 end of the molecule having no monosaccharide attached would be expected to migrate in the region of free PPEADP (fig. 12) but was not detected in the presence of the excess lipid. Oligosaccharide 01 gave rise to two neoglycolipid bands (Fig. 12) consistent with its known structure (Table 1). The first at position 1 was identical to that derived from oligosaccharide Rl with respect to mobility on TLC, chemical staining and LSIMS spectrum (Table 2); the second band at position 2 , was stained by primulin only but not orcinol, gave a molecular ion [M-H] m/z 937.6 and a prominent fragment ion m/z 734 arising from loss of GlcNAc consistent with the derivative GlcNacβl-6-OT (Table 2). Oligosaccharide fraction Nl gave rise to two major neoglycolipid bands (Fig. 12), in accord with the structure proposed (Table 1), and several minor bands which were not investigated further. The faster migrating band at position 1 had a spectrum identifying it as Galβl-3-OX (Table 2). The other major band at position 8 stained with both orcinol and primulin and gave a molecular ion [M-H] m/z 1099.7 and fragment ions m/z 937 and 734 arising from the loss of Gal and Gal- GlcNAc, respectively, consistent with the derivative Galβl- 4GlcNAcβl-6-OT. Penta- and hexasaccharide fractions: Despite the three chromatographic steps used in the fractionation of the penta- and hexasaccharides (Bio-Gel P4, reverse and normal phase HPLC), multiple components were present in several of the fractions as deduced from MS and NMR analysis [3]. TLC of the free oligosaccharides (Fig. 11) shows that these components are difficult to resolve. However they are mostly separated as neoglycolipid derivatives TE SHEET - 25 - (Fig. 13 panel A), thus allowing analysis of the individual components by LSIMS. These are discussed below in the order shown in the chromatogram except for the previously uncharacterised fraction K3(2) which was placed in the middle of the chromatogram for comparison, and is discussed separately. Fraction K5(3) gave three major neoglycolipid bands on TLC analysis, each stained by orcinol (not shown) and primulin (Fig. 13). The slowest migrating and most intense of these at position 8 had a negative ion spectrum corresponding to the sequence Galβl-4GlcNAcβl-6-0Y (Table 2), which is the predicted derivative from the 6-linked branch common to the two major oligosaccharide alditols in this fraction: Galβl-4GlcNAcβl χ 6 GalNAcol / 3 Galβl-4GlcNAcβl Galβl-4GlcNAcβl Gal NAcol 3 Galβl-3GlcNAcβl The two faster migrating bands at positions 6 and 7 each gave spectra (summarised in Table 2) consistent with the Gal-GlcNAc-3-OX sequence with [M-H] ions m/z 1200.7 and fragment ions m/z 1038 and 835 due to loss of Gal and Gal- GlcNAc, respectively. Based on the chromatographic mobilities of neoglycolipids derived from the two chemically εynthesised trisaccharide standards 03 and 07 having the Galβl-3GlcNAc and Galβl-4GlcNAc sequence linked βl-3 to Gal (Fig. 13, panel B) it is deduced that the band at position 6 is Galβl-3GlcNAcβ/-3-OX and that at position 7 is Galβl-4GlcNAcβl-3-0X. The relative amounts of the three bands given by the intensity of the orcinol and primulin stains are consistent with the previous NMR and MS data of the intact oligosaccharides which indicated that approximately equal amounts of the two isomers were present. - 26 - Fraction K2(3) gave three major neoglycolipid bands at positions 1, 8 and 9 (Fig. 13 panel A). The fastest migrating was identified as Galβl-3-OX from its negative ion spectrum (Table 2); this is the predicted derivative from the 3-linked branch of the major oligosaccharide in this fraction (Table 1). The band at position 9, gave a spectrum (Table 2) with molecular ion [M- H] ~ m/z 1302.8 and fragment ions m/z 1099, 937 and 734 consistent with the sequence GlcNAcctl-4Galβl-4GLcNAcβl-6-OY derived from the 6-linked branch of the major oligosaccharide (Table 1). In the other major band at position 8, two sequences were identified by LSIMS having [M-H]~ m/z 1099.7 and 1200.7 (Table 2). The former corresponds to the derivative Gal-GlcNAc-6-OY identified above and in the presence of Gal-3-OX at position 1 would be derived from the minor contaminant oligosaccharide Nl, previously detected (Table 1). The ion m/z 1200.7 in the presence of a fragment ion m/z 997 is suggested to arise from the derivative GlcNAc-Gal-3-OX (although the Gal- GlcNAc-3-OX sequence remains an alternative possibility in the absence of sequence ions). The relative amounts of the -6-0Y and -3-0X sequences detected in this band suggest the presence of a previously undetected pentasaccharide with the structure: Gal-GlcNAc 6 GalNAcol 3 / GlcNAc-Gal (Gal-GlcNAc) Two minor bands at positions 2 and 10, identified by LSIMS as GlcNAc-6-OY and Gal-GlcNAc-Gal-3-OX, respectively (Table 2), together account for the second minor component previously identified in fraction K2(3) (Table 1). Fraction K3(4) gave two major bands of which the faster migrating was at position 8 and identified as Galβl-4GlcNAcβl-6-OY (Table 2) and the slower, at position 10 gave an LSIMS spectrum (Table 2) with molecular ion [M-H] " m/z - 27 - 1362.8 and fragment ions m/z 1200, 997 and 835 consistent with the sequence Galβl-4GlcNAcβl-3Galβl-3-OX. The identification of these products was important in assigning the structure of the major oligosaccharide in this fraction (Table 1) in conjunction with MS and NMR data on the free alditols [3]. The minor neoglycolipid bands at position 1 and 12 having [M-H] 997.6 and 1464.8 are consistent with the presence of oligosaccharides related to Nl and to the major component of fraction K4(3) as detected previously (Table 1). A third minor band at position 13 with [M-H] m/z 1727.9 may arise from the linear sequence 13a (Table 2) in the absence of GC-MS data for the branched Gal residue in this fraction [3]. Fraction K6(2) gave one prominent neoglycolipid band at position 11 (Fig.13) with [M-H] m/z 1565.9 and fragment ions m/z 1403, 1200, 1038 and 835 consistent with the sequence Gal β l-3GlcNAcβl-3Galβl-4GlcNAc-3-OX (Table 2) which would arise from the previously identified major oligosaccharide in this fraction (Table 1). A minor band at position 12 had a negative ion spectrum identical to that from band 11 and was deduced to arise from the minor oligosaccharide isomer, position 12 structure a, previously identified (Table 1). Additional bands chromatographing at positions 7 and 8, and having LSIMS spectra identical to those derived from fraction K5(3) (Table 2) were indicative of the presence of the second pentasaccharide isomer in fraction K5(3) described above; this had not been detected in the previous NMR and MS analysis of K6(2) due to the low amounts of this fraction available. Fraction K6(3) gave two major neoglycolipid bands (Fig. 13 Panel A) of which the slower migrating band at position 9 gave a LSIMS spectrum identical to that derived from fraction K2(3) (Table 2) consistent with the structure proposed (Table 1) for the 6-linked branch of the major oligosaccharide alditol in this fraction. The faster migrating band at position 3 was shown by LSIMS (Table 2) to correspond to derivative (Fucd - 2Galβl-3-0X) from the - 28 - proposed 3-linked branch giving a molecular ion [M-H] m/z 1143.7 and fragment ions m/z 997 and 835 from loss of Fuc and Fuc-Gal respectively. This band could further be distinguished by an orange tinge given by the orcinol stain indicating the presence of fucose as a major component. Minor neoglycolipid bands at positions 7, 8 7 11 and 12 suggested the presence of oligosaccharides related to the major and minor pentasaccharides (Table 1) in fraction K6(2). Fraction K2(4) gave rise to two neoglycolipid bands: one migrated at position 8 and gabe a negative ion spectrum (Table 2) consistent with the sequence Galβl-4GlcNAcβl-6-OY that is predicted from the 6-linked branch of the major oligosaccharide in this fraction (Table 1). The second band at position 9 gave a spectrum (Table 2) having a molecular ion [M-H] m/z 1462.8 with fragment ions m/z 1200, 997 and 835 which would arise from loss of Gal, Gal-GlcNAc and Gal-GlcNAc-Gal from the derivative Galβl-4GlcNAcβl-6Galβl-3-OX. This latter sequence having an internal -6Gal has a slightly faster migration than the equivalent structure having the Galβl- 4GlcNAcβl-3Galβl-3-OX sequence as in K3(4) at position 10. Fraction K4(2) gave two prominent neoglycolipid bands migrating at positions 4 and 9. The latter band gave a negative ion spectrum identical to the derivative from fraction K2(3) (Table 2) which is the expected derivative from the 6-linked branch of the major oligosaccharide characterised previously in this fraction (Table 1). The band at position 4 gave a spectrum (Table 2) having a molecular ion [M-H] m/z 1200.5 and fragment ions m/z 997 and 835 from loss of GlcNAc and GlcNAc-Gal respectively, consistent with the sequent GlcNAccl-4Galβl-3-OX expected from the 3-linked branch of this oligosaccharide (Table 1) . The several trace neoglycolipid products were not identified. Fraction K4(3) was shown previously to contain several oligosaccharides of which only the major component was confidently characterised (Table 1). - 29 - Two of the major neoglycolipid products were consistent with the structure proposed for this oligosaccharide: the band at position 1 gave a negative ion spectrum corresponding to the sequence Galβl-3-OX, and the band at position 12 gave a spectrum (Table 2) having molecular ion [M-H] m/z 1464.8, with the fragment ions m/z 1302, 1099 and 937 resulting from loss of Gal, Gal-GlcNAc and Gal-GlcNAc-Gal consistent with the sequence 12b given in Table 2: Galβl-3GlcNAcβl-3Galβl-4GlcNAcβl-6-OY. LSIMS analysis of the prominent slower migrating band at position 13 enabled a second oligosaccharide sequence to be identified in this fraction: Gal-GlcNAc N6 Gal-3GalNAcol 3 / Gal-GlcNAc This was deduced from the LSIMS spectrum with [M-H] m/z 1727.9 consistent with the sequence (Gal-GlcNAc) z -Gal-3-OX, and the presence of only three fragment ions m/z 1565, 1362 and 835 that would arise from loss of Gal, GlcNAc-Gal and (Gal-GlcNAc) a -Gal, respectively, from either of two branches at an internal galactose residue or the entire branched structure with internal galactose (Table 2) . Confirmation of these as fragment ions rather than molecular ions of smaller contaminant oligosaccharides was given by the characteristic satellite peaks at -18 and +28 daltons from the fragment peak. These results extend data from the previous study which showed the presence of branched galactose and 3-substituted N- acetylgalactosaminitol [3] but alone could not give an unambiguous assignment of the structure of this component. Additional oligosaccharides suggested to be present from MS analysis of free oligosaccharides could be identified by LSIMS analysis of the minor neoglycolipid bands migrating at positions 4, 8, 9 and 10 (Table 2). These were hexasaccharides related to the major components in fractions K2(4), K3(4) and K4(2), deduced from the band pairs at positions 8a and 9b, 8a and - 30 - 10a and at positions 4 and 9a, respectively. Derivatives from a fourth minor oligosaccharide related to K2(3) suggested by MS analysis of the free oligosaccharides, would be contained within neoglycolipid bands at positions 1 and 9a. Previously uncharacterised fraction K3(2): This fraction consisted mainly of pentasaccharides as deduced by LSIMS analysis of the native fraction which gave only one molecular ion species [M-H] m/z 952. However the fraction was too heterogeneous to be characterised by the previous MS and NMR analyses except for the following: HI proton signals at 4.852 and 4.853 ppm (at 285K) indicated the presence of non- reducing terminal GlcNAcc residues and major H2 and H5 signals from GalNAcol were within 0.006 ppm upfield of those for oligosaccharide K2(3) indicating that the major oligosaccharides were branched with GlcNAc at C6 and Gal at C3 of the core region GalNAcol. The neoglycolipids from fraction K3(2) resolved into 6 discrete bands at positions 1, 2, 5, 8, 9 and 10 and a broad band at position 12. The bands at positions 1 and 2 were identified (Table 2) from their LSIMS spectra as the derivatives Galβl-3-OX and GlcNAcβl-6-OY. The weak band at position 5 gave a LSIMS spectrum consistent with HexNAc-Hex-3-OX from the [M-H] ion m/z 1200.7 and fragment ions m/z 997 and 835 given by loss of HexNAc and HexNAc-Hex respectively (Table 2). The slower migration of this band compared to that at position 4 (GlcNAcc(l-4Galβl-3-OX) indicates that alternative linkages or a monosaccharide other than GlcNAc were present. For the band at position 8 two sequences were found (Table 2); the first was identified as Gal-GlcNAc-6-OY with a negative ion spectrum similar to that for the band at the same position derived from oligosaccharide Nl (Table 2), and the second sequence HexNAc- Hex-3-OX, with a spectrum similar to that for the band at - 31 - position 5, but a different TLC migration. The bands at positions 9 and 10 each give a spectrum consistent with the presence of two sequences; the first GlcNAc-Gal-GlcNAc-6-OY having [M-H] m/z 1302.9, with fragment ions m/z 1099 and 937 corresponding to loss of GlcNAc and GlcNAc-Gal respectively, and the second Gal-GlcNAc-Gal-3-OX having [M-H] m/z 1362.8 and fragment ions 1200 and 997 corresponding to loss of Gal and Gal-GlcNAc respectively (Table 2). The band at position 12 gave a spectrum (Fig. 18b) consistent with a major component HexNAc- HexNAc-Hex-3-OX having [M-H] m/z 1565.9 and fragment ions 1362, 1200, 997 and 835 resulting from loss of HexNAc, HexNAc-Hex, HexNAc-Hex-HexNAc and HexNAc-Hex-HexNAc-Hex, respectively, and a minor component with the sequence Fuc-Hex-HexNAC-Hex-3-OX ([M- H] ~ m/z 1508.9). The neoglycolipid profiles and knowledge that the majority of the oligosaccharide alditols present in fraction K3(2) are pentasaccharides of mass 953 that are 3,6 branched at the GalNAcol core, suggest the presence of the following oligosaccharide structures in this fraction: derivative position 8 Gal-GlcNAcβl ^GalNAcol 5,8 HexNAc-Hexl 12 HexNAc-Hex-HexNAc-Hex-3GalNAcol - 32 - REFERENCE LIS.T FOK. £)<A πPL& PβLT{ϊή (1) Yamashita, K. , Mizouchi, T. and Kobata, A. (1982) Methods in Enzymology., 83, 105-126. (2) Hounsell, E.F. and Feizi, T. (1982) Medical Biology, 60, 227-236. (3) Hounsell, E.F., Lawson, A.M., Stoll, M.S., Kane, D.P., Cashmore, G.C., Carruthers, R.A. , Feeney, J. and Feizi, T. (1989) Eur. J. Biochem., In press. (4) Tang, P.W. , Gooi, H.C., Hardy, M. , Lee, Y.C. and Feizi, T. (1985) Biochem. Biophys. Res. Commun., 132, 474-480. (5) Lawson, A.M., Hounsell, E.F. , Wilson, S.B., Cashmore, G.C., Stoll. , M.S. and Feizi, T. (1988) Proc. 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA. (6) Hounsell, E.F., Lawson, A.M., Feeney, J., Kane, D., Cashmore, G.C., Stoll, M.S. and Feizi, T. (1988) Proc. XlVth Int. Carbohydr. Symposium (Stockholm) A92. (7) Hounsell, E.F., Lawson, A.M., Feeney, J., Gooi, H.C., Pickering, N.J., Stoll, M.S., Lui, S.C. and Feizi, T. (1985) Eur. J. Biochem., 148, 367-377. (8) Stoll, M.S., Mizouchi, T., Childs, R.A. and Feizi, T. (1988) Biochem. J., 256, 661-664. (9) Lawson, A.M., Chai, W. , Cashmore, G.C., Stoll, M.S., Hounsell, E.F. and Feizi, T. (1989) Carbohydr. Res. in press. (10) Gooi, H.C., Veyrieres, A., Alais, J., Scudder, P., Hounsell, E.F. and Feizi, T. (1984) Molec. Immunol., 21, 1099-1104. (11) Lemieux, R.U., Wong, T.C., Liao, J. and Kabat, E.A. (1984) Molec. Immunol., 21, 751-759. (12) Clancy, M.J. and Whelan, W.J. (1959) Chemistry and Industry, P673-675. (13) Mizouchi, T. , Loveless, R.W. , Lawson, A.M., Chai, W. , Lachman, P.J., Childs, R.A. , Thiel, S. and Feizi, T. (1989) J. Biol. Chem., 264, 13834-13839. - 32a - (14) Rosenstein, I.J., Stoll, M.S., Mizouchi, T., Childs, R.A. , Hounsell, E.F. and Feizi, T. (1988) The Lancet, 1327- 1330. (15) Childs, R.A., Drickamer, K. , Kawasaki, T. , Thiel, S. and Feizi, T. (1989) Biochem. J. , 262, 131-138. TABLE 1 Structures identified in the di- to hexasaccharide fractions from studies of free oligosaccharide alditols ' Taken from re s 3 and 6 assignments of the oligosaccharide structures indicated by asteri wholely dependant on TLC-MS analyses of their neoglycolipids. Fraction Major A Designations oligosaccharides c O Rl Galβl-3GalNAcol 0 01 GlcNAcβl. O IGalNΛcol Galβl / Nl GaIβl-3GlcNAcβl χ "GalNAcol Rl and 01 o Galβl /' K5(3) Galβl -4GlcNAcβl N "GalNAcol Gal-GlcNAc-Gal- Galβl-3/4GlcNAcβl ' Nl and G Galβl-3/ iGlcNAc K2(3) GlcNΛcαl-4Galβl-4GlcNAcβl χ "GalNAcol Gal- Galβl' GlcN (Gal- K2(A) Galβl-4GlcNAcβl GlcNAc-Gal-(Fu e 2 IGalNΛcol Galβl-4GlcNΛcβl-6Galβl ' Structure relate Fraction Major Addit Designations oligosaccharides compo K3(4) Galβl -4GlcNAcβl v ^GalNAcol* Structures relat Galβl --**GlcNAcβl-3Galβl / K6(2) Galβl-3GlcNAcβl-3Galβl -4GlcNAcβl ~3GalNAcol Galβl-3GlcNAcβl- and K5(3)>|<. K6(3) GlcNAcαl- iGalβl -4GlcNAcβl v Structures rela ,GalNAcol and KSO)^ Fucαl-2Galβl' 4 K4 (2) GlcNAcαl-AGalβl--4GlcNAcβl χ FuclGal.GlcNAcJ ,GalNAcol GlcNAcαl-AGalβl / Structures rela I K2(A), K3(4) an K4(3) Galβl-3GlcNAcβl-3Galβl -«GlcNAcβl v Galβl-3/ GlcNAc ,GalNΛcol Galβl' Galβl -3/AGlcNAc TABLE 1 (CONTINUED) TABLE 2 LSIMS data and structures of neoglycolipids derived from oligosaccharide alditol fractions. X=όH 2 (-ύH-MlAc-cH 2 όH)-NH-CH 2 -όH 2 -ό-P(-ό)(-OH)-0-όH 2 -K>; Y = CH 2 -CH 2 -NH-CH 2 -CH 2 -NH-CH 2 -CH 2 -0-P(-0)(-0H)-0-CH 2 -DP where DP = -CH(CHOCO(CH 2 ) 1/ .CH 2 )-OCO(CH 2 ) 14 CH 2 . Asterisks indicate instances where molecula ions only were observed. Fragment ion masses are rounded down to Integral values. O TLC Mass of Mass of Oligosaccharide O Position Structure" GaJ.βl-3-OX ClcNAcβl-6-OY i rβcol-2Galβl -3-OX o GlcNΛcαl-4Galβl-3-OX H«_j*_NΛc-Heχ-3-0X 1200.7 997,835 K3(2) 6 Calβl -3GlcNAcβl -3-OX 7 Galβl-4GlcNΛcβl -3-OX 8a G»lβl -4GlcNΛcβl-6-OY 8b HexNAc -Hex -3 -OX 9a GlcNΛcαl -4Galβl -4ClcNAcβl-6-OY 9b Galβl -4GlcNΛcβl -6Galβl-3-OX TLC Mass of Mass of Oligosaccharide Position Structure (M-H)~ ion fragment iohs alditol fractions 10a Galβl-4GlcNΛcβl -3Galβl-3-0X - 1362.8 1200,997,835 2(3)*,K3(2),K3(4), K4(3)* 10b GlcNAc-Gal-GlcNAc-6-OY 1302.8 1099,937 3(2) O 11 Galβl-3GlcNΛcβl-3Calβl-4GlcNΛcβl-3-0X 1565.9 1403.1200,1038,835 K6(2) 12a Galβl -3GlcNΛcβl -3Galβl -3GlcNAcβl -3-0X 1565.9 K6(2)*,K6(3)* 12b Galβl-3GlcNΛcβl-3Galβl-4GlcNAcβl -6-0Y 1464.8 1302,1099,937 K3(4),K4(3) 12c HexNAc-Hex-HexNAc-Hex-3-OX 1565.9 1362,1200,997,835 3 ) 12d Fuc-Hex-HexNAc-Hex-3-OX 1508.9. K3(2)* 13a Ga βl-3/4GlcNAcβl-3Galβl-3/4GlcNAcβl-3Galβl-30X 1727.9 1362.1200, 997, 835 K3(4) 13b Calβl-3/4GlcNAcβl χ 1727.9 ,Galβl-3-0X 1565.1362,835 K4(3) Gal31-3/4GlcNAcβl TABLE 2 (CONTINUED)

Claims: CLAIMS 1. A method of separating 0-linked and/or N-linked oligosaccharide ' groups from a sample comprising glycoprotein, proteoglycan or glycopeptide to which said groups are linked, wherein said groups are removed from said glycoprotein, proteoglycan or glycopeptide wi-thϊα the sample and subsequently, in their alditol form, caused to contact a support bearing a reactive dihydroxyboronyl group to selectively bind said alditol oligosaccharide groups in the absence of binding other groups to said support and thereafter removing the bound groups from the support. 2. A method as claimed in claim 1 wherein O-linked and N-linked oligosaccharide groups are sequentially separated. 3. A method as claimed in claim 1 or 2 wherein the protein -proximal monosaccharide of O-linked oligosaccharides removed is additionally selectively cleaved at its C4-C ' linkage by the action ' of a mild oxidising agent, such as periodate preferably including a quenching agent. 4. A method as claimed in claim 3 wherein the selective oxidation cleavage is followed by a coupling reaction to amino-lipid molecules or other amino- containing molecule. 5. A method as claimed in any preceding claim as applied to the separation of oligosaccharide groups from mucin-type glycoprotein. 6. A method as claimed in any preceding claim wherein the support comprises a phenylboronic acid column. 7. A method of separating O-linked oligosaccharide groups from a glycoprotein or peptide sample containing O-linked and/or N-linked oligosaccharide groups which comprises (a) removing O-linked oligosaccharides from glycoprotein or peptide to which they are bound in a given sample, and converting the oligosaccharides to alditol form; (b) passing the sample through a phenylboronic acid (PBA) column to retain formerly O-linked oligosaccharides thereon, and (c) eluting the column under acidic conditions to release said formerly O-linked oligosaccharides. 8. A method as claimed in claim 7 further comprising (d) treating the fall through obtained in step (b) to release N-linked oligosaccharides; (e) reduction of N-linked oligosaccharides to their alditol form, (f) passing material obtained from step (e) through a phenylboronic acid column such that the oligosaccharide alditols are retained thereon, and (g) eluting the column under acidic conditions to release said alditols. 9. A method as claimed in claim 7 or 8 wherein a fluorescent or radioactive label is introduced to the oligosaccharide groups prior to step (b). 10. A method as claimed in any one of claims 7 to 9 including sequential separation of O-linked and N-linked oligosaccharide groups. 11. A method as claimed in any one of claims 7 to 10 wherein the terminal (protein-proximal or peptide-proximal) monosaccharide unit in _ oligosaccharide is selectively - 3 - cleaved at its C4-C5 linkage under the action of a mild oxidising agent. 12. A method as claimed in claim 10 wherein the selective oxidative cleavage is followed by a coupling reaction to amino-lipid molecules or other amino- containing molecule. 13. A method as claimed in any one of claims 7 to 12 applied to the separation of O-linked and/or N-linked oligosaccharide groups from mucin-type glycoprotein. 14. A method as claimed in any preceding claim wherein the sample is applied to the support or column in alkaline pH and said column or support is subsequently eluted under acidic pH. 15. A kit suitable for carrying into effect a method as claimed in any preceding claim comprising a support having reactive dihydroxyboryl groups together with an analytical standard or standards identifying specific oligosaccharide derivatives or fragments. 16. Apparatus suitable for carrying into effect a method as claimed in any one of claims 1 to 14. 17. A method of selectively cleaving the C4-C5 bond of the terminal (protein- or peptide-proximal) monosaccharide moiety of an oligosaccharide alditol by use of a mild oxidising agent. 18. A method as claimed in claim 17 wherein the oxidising agent comprises periodate. 19. A method as claimed in claim 17 or 18 carried out in the presence of a quencing agent to inactivate excess oxidant.

Inventors: FEIZI TEN, HOUNSELL ELIZABETH FAY, LAWSON ALEXANDER MACNAIR, STOLL MARK SEBASTIAN, FEIZI, TEN, HOUNSELL, ELIZABETH, FAY, LAWSON, ALEXANDER, MACNAIR, STOLL, MARK, SEBASTIAN
Applicants: 3I RESEARCH EXPLOIT LTD
Application year: 1989
Main classification: C07H1
IPC codes: C07H1, C07H13, C07H15, G01N30, G01N33