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Glycoprotein methods protocols - biotechnology
O-Linked Chain Release and Fractionation 18118115O-Linked Oligosaccharide Chain Releaseand FractionationElizabeth F. Hounsell1. IntroductionO-linked chains of glycoproteins have classically been released by alkaline boro-hydride degradation, in which mild alkali (0.05 M OH–) is used to cause β-eliminationfrom the β carbon of serine (R-H) or threonine (R-CH3) in the protein backbone.To inhibit subsequent elimination around the glycosidic ring of the linkage monosac-charide, and on backward down the oligosaccharide if linked at C-3, the reaction iscarried out in the presence of 1 M sodium borohydride to give simultaneous reductionof the reducing sugar formed after elimination. An advantage of this technique is thatthere is now a large database of nuclear magnetic resonance (NMR) chemical shiftsfor mucin-type oligosaccharide alditols (1,2) that can be searched in a computer-as-sisted way (3) for structural identification. The disadvantage of this technique is thatthe resulting alditol is not capable of undergoing a reductive amination procedure forcoupling to a sensitive flourescent label or for polyvalent coupling to lipid or proteinfor subsequent immunoassay. However, it is possible to reoxidize selectively thealditol using periodate, which results in a new aldehyde being formed for reductiveamination, and, indeed, this is the basis for a method for structural analysis by thin-layer chromatography-mass spectometry (MS) (4). Note, however, that for subsequentbiological or immunological assay, any branching at GalNAc-Ser/Thr, often found inmucins, is destroyed by this procedure.From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 182 HounsellIf branching is not present, a useful cleavage can be obtained by the enzyme O-glycanase (α-N-acetylgalactosaminidase). This enzyme has the disadvantage that onlyrelatively simple oligosaccharides are released with any reproducibility (5,6); the dis-accharide Galβ1-3GalNAcα1-Ser/Thr is the best substrate. Hydrazinolysis is prob-ably the preferred technique for release of all oligosaccharides in both the presenceand the absence of sulfate and/or sialic acids, if the conditions are optimized. It may bea good idea to carry out this reaction in triplicate with the alkaline borohydride degra-dation and O-glycanase in order to ensure that the complete picture of a mucin at astructural level is obtained. Note that if the protein is required for analysis, rather thanthe oligosaccharide, mild alkali in the absence of borohydride (7) or hydrolysis withtrifluoromethanesulfonic acid (TFMSA) can be used to keep the protein intact.After the release of oligosaccharide alditols, these can be fractionated by normal- or re-versed-phase high-performance liquid chromatography (HPLC) (8) or anion-exchange chro-matography, e.g., high-performance anion-exchange chromatography (HPAEC) on twoCarboPac PA-100 columns (Dionex Camberley, Surrey, UK) in series (9). Sulfated andsialylated oligosaccharide alditols can also be separated by these techniques; however, forbetter results the reversed-phase HPLC should be by porous graphitized carbon (PGC) in0.1% trifluoroacetic acid (TFA) (10,11), and the normal-phase HPLC should be in the pres-ence of buffers (9). Sulfated oligosaccharide alditols are retained well on the HPLC columns(12). For reducing oligosaccharides, PGC is again a good alternative for both neutral andanionic oligosaccharides, which have similar retention times to each other and to their alditols.On HPAEC (13), the reducing oligosaccharides will be retained significantly longer than thealditols, to give improved chromatography. Sulfated oligosaccharides may now be retainedtoo long on a CarboPac PA-100 column. Although HPAEC with pulsed amperometric detec-tion (PAD) is a sensitive technique, additional sensitivity can be obtained by flourescentlabeling. The usual labels, 2-aminopyridine (14,15) and 2-aminobenzamide (15,16) causesulfated oligosaccharides to be retained too long on reversed phase or PGC column. Forneutral or sialylated oligosaccharides, separation is primarily achieved by reversed-phaseand normal-phase chromatography, often as a two-dimentional map (14,15), or on weak ionexchange, e.g., GlycoSep C™ (Oxford GlycoSciences, Abingdon, Oxford, UK) (17). Neu-tral 2-aminobenzamide (2-AB) oligosaccharides (naturally occuring or after desialylation)can be analyzed by gel filtration in water as eluent (usually Bio-Gel P4 chromatography) togive molecular size estimation. This is particularly useful to follow exoglycosidase diges-tions to obtain additional sequence information. More information about these techniquescan be obtained from refs. 18–20. The use of electrospray ionization MS coupled with colli-sion-induced dissociation MS is another possibility for analysis at the glycopeptide level(21). The internet provides a source for predicting potential O-glycosylation sites on proteinsat http://www.cbs.dtu.dk/netOglyc/cbsnetOglyc.html.2. Materials2.1. Alkaline Borohydride Degradation (β-Elimination)and Chromatography1. 1 M NaBH4 in 0.05 M NaOH made up fresh.2. Glacial acetic acid. O-Linked Chain Release and Fractionation 1833. Methanol.4. Cation-exchange column Dowex 50W X8 H+ form.5. Phenyl boronic acid (PBA) Bond Elut columns (Jones Chromatography, Hengoed, UK)activated with MeOH (22).6. 0.2 M NH4OH.7. 0.01, 0.1, and 0.5 M HCl.8. HPLC apparatus fitted with an ultraviolet detector (approx 1 nmol of mono- and oligosac-charides containing N-acetyl groups can be detected at 195–210 nm) and pulsed electro-chemical detector (oligo- and monosaccharides ionized at high pH can be detected atpicomole level).9. Columns: reversed-phase C18, amino bonded silica, PGC (Hypercarb 7µ, Hypersil Ltd.,Runcorn Cheshire, UK), CarboPac PA100 and CarboPac PA1 (Dionex Camberley).10. Eluents for HPLC: HPLC grade water, acetonitrile, 0.1% aqueous TFA; acetonitrile con-taining 0.1% TFA, ammonium formate.11. Eluents for HPAEC: 12.5 M NaOH (BDH, Poole, Dorset, UK) diluted fresh each day to200, 100, 80, and 1.5 mM. After chromatography and detection, salt needs to be removedby a Dionex micromembrane suppressor or by cation-exchange chromatography beforefurther analysis, e.g., by methylation.12. Eluent A: 0.08 M NaOH.13. Eluent B: 0.5 M sodium acetate (Aldrich, Poole, Dorset, UK) in 0.08 M NaOH.2.2. Reoxidation1. Sodium periodate, analytical reagent grade.2. Imidazole (Sigma, Poole, Dorset, UK) 40 mM adjusted to pH6.5 with HCl3. Butan-2,3-diol.2.3. Release of the Core 1 Disaccharide (Galβ1-3GalNAcα–) from Mucins1. Bio-Spin®chromatography columns (Bio-Rad, Hercules CA), or home made columnssuitable for 1.5-mL microcentrifuge tubes packed with 0.8 mL of Bio-Gel P30 acrylamidegel matrix . Store at 4°C in 0.15 M sodium chloride-17.5 mM sodium citrate, pH 7.0,containing 0.2% w/v sodium azide as preservative.2. O-Glycanase®; Streptococcus (Diplococcus) pneumoniae Endo-α-N-acetylgalacto-saminidase (EC 3.2.1.907) (Oxford GlycoSciences). Store at –20°C for up to 6 mo, butavoid repeated freeze-thawing.3. 0.1 M sodium citrate-phosphate, pH 6.0; make up with HPLC-grade water.4. Dowex 50X-12 H+-form resin.5. Sephadex GM25.2.4. Hydrazinolysis1. 0.5-mL screw-capped V-bottomed Reacti-vials™ (Pierce and Warriner, Chester, UK).2. Anhydrous hydrazine (Pierce and Warriner).3. Whatman CF-11 cellulose chromatography medium.4. Reagent A: butanol:ethanol:acetic acid, 4:1:0.5 (v/v/v).5. Reagent B: butanol:ethanol:water, 4:1:1 (v/v/v).6. Reagent C: acetic anhydride:methanol, 2:5 (v/v).7. Reagent D: 0.2 M sodium acetate.8. Sep-Pak C18 cartridge (Waters, Watford, UK).9. Alternatively, the Glycorelease N- and O-Glycan recovery kit (Oxford GlycoSciences)can be used. 184 Hounsell2.5. Isolation of the Protein by TFMSA Destruction of Oligosaccharides1. Anhydrous TFMSA (23).2. Reagents as specified in Glyco Free™ Deglycosylation kit (K500, Oxford, Glyco-Sciences).2.6. 2-AB Fluorescence Labeling and Size ExclusionChromatography (SEC)1. Signal labeling kit (K404,Oxford GlycoSciences).2. Dowex AG50 X 12 (H+ form).3. Dowex AG1 X 8 (acetate form).4. RAAM 2000 Glycosequencer (Oxford GlycoSciences) or Biogel P4 column (100 × 2 cm)in a water jacket at 55°C and HPLC pump with refractive index and fluorescence detectors.5. GlycoSepH™ and GlycoSepC™ HPLC column (Oxford GlycoSciences).3. Methods3.1. Alkaline Borohydride Degradation (β-Elimination)1. Release O-linked chains by treatment with 0.05 M sodium hydroxide in the presence of 1 MNaBH4 or NaB[3H]4 for 16 h at 50°C.2. Degrade excess NaBH4or NaB[3H]4by the careful addition with the sample on ice ofglacial acetic acid (to pH 7.0) or acetone (1 mL/100 mg of NaBH4) followed by repeatedevaporation with methanol.3. Desalt on a cation-exchange column and analyze by reversed-phase HPLC (Subheading3.6.2.) or HPAEC (Subheading 3.7.1.).4. Or, for microscale identification of the presence of alditols, dissolve the sample in 200 µL0.2 M NH4OH and add to the top of a PBA minicolumn prewashed with MeOH, water,and 0.2 M NH4OH.5. Wash the PBA column with 2 × 100 µL 0.2 M of NH4OH and 2 × 100 µL of water.6. Specifically elute the alditols in 1 M acetic acid.7. Evaporate the sample and reevaporate with 2 × 100 µL of water.3.2. Oxidation of Oligosaccharide Alditols1. To the dry alditols, add twice the molar ratio of sodium periodate in imidazole buffer (seeNote 1).2. Oxidize in the dark at 0°C for 5 min.3. Destroy excess oxidant with butan-2,3-diol (two times the molar excess over periodate)for 40 min at 0°C in the dark.3.3. Release of Core 1 Disaccharide (Galβ1-3GalNAcα–) from MucinUsing Endo-α-N-Acetylgalactosaminidase (O-Glycanase)3.3.1. The Removal of Glycerol from O-Glycanase (see Note 2)1. Invert a Bio-Spin P30 polyacrylamide BioGel column (0.8 mL column volume) severaltimes and allow the buffer to drain by gravity.2. Wash the column with 300 µL of 0.01 M citrate-phosphate, pH 6.0, place in a collectiontube, and centrifuge for 2 min at 1100g. Repeat four times.3. Make up 6 mU O-Glycanase to 100 µL with 0.01 M citrate-phosphate, pH 6.0, and loadthe sample carefully and directly to the center of the column, drop wise (see Note 3).4. Centrifuge for 4 min at 1100g and collect the excluded O-Glycanase. O-Linked Chain Release and Fractionation 1855. Pass the excluded O-Glycanase through a second Bio-Spin column to maximize glycerolremoval.3.3.2. Hydrolysis of Galβ1-3GalNAcα- from mucin using O-Glycanase1. Reconstitute mucin (100 µg) with 90 µL of 0.1 M citrate-phosphate, pH 6.0, containing100 µg/mL of bovine serum albumin and 0.02% (w/v) sodium azide. Mix well.2. Add 0.6 mU of deglycerolyated O-Glycanase (10 µL).3. Incubate for 18 h at 37°C (see Note 4).4. Load reactions on Dowex 50X-12H+form resin (3 × 0.5 cm column) and elute with threecolumn vol of HPLC-grade water.5. Collect the effluent and eluent and then pool (2.5-mL volume).6. Load onto a PD10-Sephadex GM25 column and elute with HPLC-grade water to isolateliberated disaccharide from intact mucin.3.4.O-Linked Oligosaccharide Release by Hydrazinolysis1. Dry salt-free glycoprotein into a V-bottomed Reacti-vial and remove from the lyophilizerimmediately before the reaction is due to commence.2. Using a clean, dry, acid-washed glass pipet, transfer 100 µL of anhydrous hydrazine tothe vial and cap immediately. Incubate at 60°C for 5 h (see Note 5).3. Allow the Reacti-vial to cool to room temperature, and transfer the reaction mixture to a1-mL cellulose (Whatman CF-11) microcolumn washed with reagent A.4. Wash the column with 3 × 1mL reagent B.5. Re-N-acetylate the glycans on the column by adding 1.4 mL of reagent C for 30 min atroom temperature.6. Wash the column with 4 × 1 mL of reagent B followed by 1 mL of methanol (see Note 6).7. Elute the oligosaccharides with 2 × 1 mL of reagent D.8. Complete the re-N-acetylation with 0.1 mL of acetic anhydride for 30 min at room tem-perature.9. Wash a Sep-Pak C18cartridge with 2 mL of methanol and 2 mL of H2O.10. Transfer the sample containing O-glycans to the cartridge and collect the eluate. Elute theremaining glycans with 0.5 mL of H2O.3.5. TFMSA Treatment1 Make up a 3 g/mL solution of TFMSA in anisole and cool in dry ice/ethanol2. Add 10 times the weight of TFMSA in anisole to the lyophilized material in a teflonscrew-capped vial standing on a bed of ice.3. Incubate at 0°C for 6–16 h with occasional vigorous shaking.4. With the vial on ice, add 1 vol of cold anhydrous diethyl ether and then add this mixtureto 1 vol of a frozen slush of aqueous pyridine.5. Warm the solution to room temperature and extract with ether.6. Collect the aqueous phase containing the peptide with partial glycosylation depending onthe reaction time.7. Alternatively, follow the instructions in the GlycoFree kit.3.6. HPLC of 2-AB-Labeled Oligosaccharides3.6.1. Preparative HPLC on a GlycoSep C HPLC Column1. Wash the column with water for 30 min at a flow rate of 0.4 mL/min.2. Wash the column with acetonitrile for 30 min at a flow rate of 0.4 mL/min. 186 Hounsell3. Wash the column with 0.5 M ammonium acetate, pH 4.5, for 30 min at a flow rate of0.4 mL/min (see Note 7).4. Rewash the column with water for 30 min at a flow rate of 0.4 mL/min.5. Equilibrate the column in 75% aqueous acetonitrile at a flow rate of 0.4 mL/min.6. Inject the 2-AB-labeled sample in 75% aqueous acetonitrile with fluorescence detectionusing an excitation λ = 330 nm and an emission λ = 420 nm.7. Elute the sample with the following gradient with fraction collection at a flow rate of0.4 mL/min (see Note 8):a. 75% acetonitrile for 5 minb. 62.5% acetonitrile over the next 25 minc. 60% over the next 30 mind. Back to 75% at 60 min for the total run3.6.2. Porous Graphitized Carbon Chromatography1. Wash the column with water for 30 min at a flow rate of 0.75 mL/min (see Note 9).2. Wash the column with acetonitrile for 30 min at a flow rate of 0.75 mL/min.3. Equilibrate the column in 0.2% TFA in 20% aqueous acetonitrile at a flow rate of0.75 mL/min (see Note 10).4. Inject the 2-AB-labeled sample in water, with fluorescence detection using an excitationλ = 330 nm and an emission of λ = 420 nm (see Note 11).5. Elute the sample with the following gradient at a flow rate of 0.75 mL/min with water/acetonitrile containing 0.2% TFA:a. 20% acetonitrile for 2 minb. 40% acetonitrile over 33 minc. 30% acetonitrile over another 35 mind. Back to 20% acetonitrile over the next 4 min6. Store the column in 75:25 (v/v) acetonitrile:water.3.7. HPAEC of Released Oligosaccharides3.7.1. HPAEC Analysis of Alkaline-Borohydride–Released MucinOligosaccharide Alditols1. Inject an aliquot (50 and 100 µL) of released mucin oligosaccharide alditols, containing320 ng of D-melibiose as internal standard, onto the two CarboPac PA-100 columns (con-nected in series) equilibrated in eluent A.2. Elute with an increasing gradient of eluent B at a flow rate of 0.7 mL/min.3. Profile the chromatogram obtained using a range of well-characterized human oligosac-charide alditol standards.4. Reequilibrate the columns before each subsequent sample application.3.7.2. HPAEC Analysis of O-Glycanase-Released Galβ1-3GalNAc1. Inject a 1-µg aliquot of the O-Glycanase-released product, containing 320 ng of D-meli-biose as internal standard, onto the two CarboPac PA100 columns (connected in series)equilibrated in eluent A.2. Elute with an increasing gradient of eluent B at a flow rate of 0.7 mL/min.3. Quantify the Galβ1-3GalNAc released using a calibration curve for standard amounts ofGalβ1-3GalNAc (see Note 12).4. Reequilibrate the column before each subsequent sample application (see Note 13). O-Linked Chain Release and Fractionation 1873.8. Fluorescence Labeling with 2-AB1. Dry salt free glycans into a 0.5-mL Eppendorf tube.2. Prepare labelling reagent of 70% DMSO 30% glacial acetic acid containing 0.25 M of 2-AB and 0.1 M of NaCNBH3(see Note 14).3. Add 5 µL of labeling reagent and incubate the sample at 65°C for 2 h.4. Centrifuge sample briefly.5. Transfer to a hydrophillic separation disk (supplied with labeling kit) washed with 1 mLof water, lml 30% acetic acid, and 1 mL of acetonitrile.6. Load the sample onto disk and leave for 15 min (see Note 15).7. Wash the tube with 100 µL acetonitrile and add to disk.8. Wash disk with 1 mL of acetonitrile followed by 5 × 1 mL 4% water in acetonitrile.9. Elute the sample with 3 × 0.5mL of water (see Note 15).10. Dry the sample to about 100 µL.11. Prepare 150 µL of AG50-X12 resin in a microcolumn and wash with 5 mL 1.5% triethy-lamine in water followed by 3 × 1 mL water (see Note 16).12. Add 150 µL of AG1 X8 (acetate form) to the microcolumn taking care not to disturb theAG50 resin.13. Wash with 0.5 mL of water.14. Load the sample in 100 µL water and elute with 4 × 0.4 mL of water.15. Filter the sample through a 0.45-µm filter and dry for further analysis.4. Notes1. Standard O-linked chains, i.e., those having the linkageare cleaved by periodate at the C-4—C-5 bond, thus giving a characteristic product forchains linked at the C-3 and/or C-6.2. The efficient removal of glycerol (added as an enzyme stabilizer) from commercial O-Gly-canase is necessary for the subsequent HPAEC analysis of hydrolyzed Galβ1-3GalNAcα-,because glycerol was found to be highly PAD active (20). Deglycerolated O-Glycanase, how-ever, does not store well, so it is best to remove the glycerol on the day of use.3. The optimum volume of sample is between 50 and 100 µL. Applying more or less resultsin poor recovery of desalted sample. Likewise, sample directed down the side of the gelalso results in poor recovery.4. 1 mU of O-glycanase releases approx 500 ng of Gaβ1-3GalNAc from 1 µg of antifreezeglycopeptides and 50 ng from 1 µg of asialofetuin (20).5. The reaction should be incubated in either an oven or a heating block but not in a waterbath. Because of the highly toxic and flammable nature of hydrazine, all manipulationsthat involve its use should be carried out in a fume cupboard with additional skin and eyeprotection. The evaporator used should also be vented into a fume cupboard; if this in-cludes a pump, the pump should be left on overnight. For the release of N-linked glycansincubate at 95°C for 5 h. 188 Hounsell6. If the column is not washed with methanol, the final eluent will consist of an immisciblebutanol/water mixture, which will prove difficult to dry.7. To ensure that high-purity eluents are always obtained, it is recommended that ammo-nium acetate be obtained by titrating the relevant acid (e.g., 0.5 M HPLC-grade aceticacid) to the relevant pH with HPLC-grade ammonium hydroxide. Ammonium formatemay also be used as an eluent, at similar pH values.8. The precise elution gradient can be varied to suit the diversity of oligosaccharides beingstudied. The pH of the ammonium acetate will greatly affect the resolution and retentionand can be tailored to suit the analytes being investigated.9. This flow rate is for a 100 × 4.6 mm Hypercarb S column. The smaller Glycosep H col-umn should not be run at flow rates in excess of 0.5 mL/min.10. Great care should be taken to ensure thorough equilibration of the column prior to injectingsamples because these columns are quite sensitive to changes in organic-phase composition.11. The use of 0.1% TFA will cause quenching of fluorescence detection.12. To optimize detector sensitivity and avoid baseline drift at low NaOH concentrations, theaddition of 0.3 M NaOH postcolumn (i.e., before it enters the detector) is required toincrease pH to ≥12. Use of a thinner electrode gasket (0.005 in.) gives the best signal-to-noise ratio. Flow rate at which the postcolumn reagent is added should be reproduciblebetween runs and must be the gradient pump flow; a pressure of 112–114 psi (eluentvessel must be able to withstand higher pressure) should achieve this flow rate with nosignificant increase in gradient pump pressure. Beware of air bubbles in the beaded mix-ing coil, which prevent efficient pH increase. Note: Always turn off the gradient pumpbefore turning off the postcolumn eluent stream (20).13. Retention times will steadily decrease unless the column is regularly regenerated withstrong alkali. Regenerate the CarboPac PA-100 anion-exchange column for 5 min with20% 0.5 M sodium acetate – 15 µM NaOH and then re-equilibriate for 30 min with 15 µMNaOH before each subsequent sample application.14. In addition to introducing 2-AB groups, reductive amination can also be used to introduceother fluorescent labels, such as 2-aminopyridine (24) or 8-amino naphtalene-1,3,6-trisulfonic acid (25), and also to couple to protein or lipid (see Subheading 1.). Here thecommercial labeling reagent is sufficient to label up tp 50 nmol of oligosaccharide. Ifpoor sloubility of the reductant (NaCNBH3) is observed, this can be improved by theaddition of 10 µL of water to the labeling mixture prior to adding it to the samples.15. Care should be taken so that the flow rate through the disk is approx 1 drop/s and that airbubbles do not form below the disk. Air bubbles can be removed by gentle pressure on thedisk; however, it is difficult to remove them completely.16. Desalting may also be carried out by Bio-Gel P2 chromatography eluted in water.AcknowledgmentThe author wishes to thank Gail Evans for preparation of the manuscript and theUK Medical Research Council for funding.References1. Hounsell, E. F. (1995) 1H-NMR in the structural and conformational analysis of oligosac-charides and glycoconjugates, in Progress in NMR Spectroscopy, vol. 27 (Emsley, J. W.,Feeney, J., and Sutcliffe, L. H., eds.), Elsevier, Kidlington, Oxford, UK, pp. 445–474. O-Linked Chain Release and Fractionation 1892. Hounsell, E. F., Davies, M. J., and Renouf, D. V. (1996) O-linked protein glycosylationstructure and function. Glycoconj. J. 13, 19–26.3. Hounsell, E. F. and Wright, D. J. (1990) Computer assisted interpretation of 1H-NMRspectra in the structural analysis of oligosaccharides. Carbohydr. Res. 205, 19–29.4. Stoll, M. S., Hounsell, E. F., Lawson, A. M., Chai, W., and Feizi, T. (1990) Microscalesequencing of O-linked oligosaccharides using periodate oxidation of alditols, coupling tophosphatidylethanolamine dipalmitoate and TLC-MS analysis of the resulting neoglyco-lipids. Eur. J. Biochem. 189, 499–507.5. Iwase, H., Ishii, I., Isihara, X., Omura, S., and Hotta, K. (1988) Release of oligosaccha-rides possessing reducing end N-acetylgalactosamiine from mucus glycoprotein in Strep-tomyces sp. OH-11242 culture medium through action of endo-type glycosidase. Biochem.Biophys. Res. Commun. 151, 4222–4228.6. Fan, J.-Q., Yamamoto, K., Matsumoto, Y., Hirabayashi, Y., Kumagai, H., and Tochikura,T. (1990) Action of endo-α-N-acetylgalactosaminidase from Alcalgenes sp on amino acid-O-glycan: comparison with the enzyme from Diplococcus pneumoniae. Biochem. Biophys.Res. Commun. 169, 751–757.7. Hounsell, E. F., Jones, N. J., and Stoll, M. S. (1985) The application of high performanceliquid chromatography to the purification of oligosaccharides containing neutral andacetamido sugars. Biochem. Soc. Trans. 13, 1061–1064.8. Hounsell, E. F. (1986) HPLC of carbohydrates, in HPLC of Small Molecules. (Lim, C. K.,ed.), IRL Press, pp. 49–68.9. Campbell, B. J., Davies, M. J., Rhodes, J. M., and Hounsell, E. F. (1993) Separation ofneutral oligosaccharide alditols from human meconium using high pH anion exchangechromatography. J. Chromatogr. 622, 137–146.10. Davies, M. J., Smith, K. D., Carruthers, R. A., Chai, W., Lawson, A. M., and Hounsell, E.F. (1993) The use of a porous graphitised carbon (PGC) column for the HPLC of oligosac-charides, alditols and glycopeptides with subsequent mass spectrometry analysis. J.Chromatogr. 646, 317–326.11. Davies, M. J. and Hounsell, E. F. (1996) Comparison of separation modes for high-perfor-mance liquid chromatography of glycoprotein- and proteoglycan-derived oligosaccharides.J. Chromatogr. 720, 227–234.12. Lo-Guidice, J-M., Herz, H., Lamblin, G., Plancke, Y., Roussel, P., and Lhermitte, M. (1997)Structures of sulfated oligosaccharides isolated from the respiratory mucins of a non-secre-tor (O, Le(a+b–)) patient suffering from chronic bronchitis. Glyco. J. 14, 113–125.13. Davies, M. J. and Hounsell, E. F. (1998) HPLC and HPAEC of oligosaccharides and glyco-peptides, in Glycoscience Protocols (Hounsell, E. F., ed.), Humana, Totowa, NJ, pp. 79–100.14. Kakehi, K., and Honda, S. (1993) Analysis of carbohydrates in glycoproteins by high-per-formance liquid chromatography and high-performance capillary electrophoresis, in Glyco-protein Analysis in Biomedicine (Hounsell, E. F., ed.), Humana, Totowa, NJ, pp. 69–80.15. Routier, F. H., Hounsell, E. F., Rudd, P. M., Takahashi, N., Bond, A., Axford, J. S., andJefferis, R. Quantitation of human IgG glycoforms isolated from rheumatoid sera: A criti-cal evaluation of chromatographic methods. J. Immunol. Methods 213, 113–130.16. Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., and Dwek, R. A. (1996) A rapid highresolution HPLC method for separating glycan mixtures and analysing oligosaccharideprofiles. Anal. Biochem. 240, 210–226.17. Guile, G. R., Wong, S. Y. C., and Dwiek, R. A. (1994) Analytical and preparative separa-tion of anionic oligosaccharides by weak anion-exchange high-performance liquid chro-matography on an inert polymer column. Anal. Biochem. 222, 231–235. 190 Hounsell18. Hounsell, E. F. (ed.) (1998) Glycoanalysis Protocols, 2nd ed., Humana Press, Totowa, NJ.19. Paramonov, N. and Hounsell, E. F. (2000) Application of HPLC to analysis of oligosac-charides of glycoproteins, in HPLC of Small Molecules, 2nd ed. (Perret, D., Flanagan, R.,and Hounsell, E. F., eds.), Oxford University Press, Oxford, UK.20. Campbell, B. J. and Rhodes, J. M. (1998) Purification of gastrointestinal mucins and analy-sis of their O-linked oligosaccharides, in Glycoanalysis Protocols, vol. 76 (Hounsell, E.F., ed.), Humana Press, Totowa, NJ.21. Greis, K. and Hart G. (1998) Analytical methods for the study of O-GlcNAc glycoproteinsand glycopeptides, in Glycoanalysis Protocols, vol. 76 (Hounsell, E. F., ed.), HumanaPress, Totowa, NJ.22. Stoll, M. S. and Hounsell, E. F. (1988) Selective purification of reduced oligosaccharidesusing a phenylboronic acid bond elute column: potential application in HPLC, massspectrometry, reductive amination procedures and antigenic/serum analysis. Biomed.Chromatogr. 2, 249–253.23. Gerken, T. A., Owens, C. L. and Pasumarthy, M. (1996) Determination of the site-specificO-glycosylation pattern of the porcine submaxillary mucin tandem repeat glycopeptide. J.Biol. Chem. 272, 9709–9719.24. Natsuka, S. and Hase S. (1998) Analysis of N– and O-Glycans by Pyridylamination, inGlycoanalysis Protocols, vol. 76 (Hounsell, E. F., ed.), Humana Press, Totowa, NJ.25. Klock, J. C. and Starr, C. M. (1998) Polyacrylamide gel electrophoresis of fluorophore-labelled carbohydrates from glycoproteins, in Glycoanalysis Protocols, vol. 76 (Hounsell,E. F., ed.), Humana Press, Totowa, NJ. [...]... Regenerate the CarboPac PA-100 anion-exchange column for 5 min with 20% 0.5 M sodium acetate – 15 µM NaOH and then re-equilibriate for 30 min with 15 µM NaOH before each subsequent sample application. 14. In addition to introducing 2-AB groups, reductive amination can also be used to introduce other fluorescent labels, such as 2-aminopyridine (24) or 8-amino naphtalene-1,3, 6- trisulfonic acid (25),... will consist of an immiscible butanol/water mixture, which will prove difficult to dry. 7. To ensure that high-purity eluents are always obtained, it is recommended that ammo- nium acetate be obtained by titrating the relevant acid (e.g., 0.5 M HPLC-grade acetic acid) to the relevant pH with HPLC-grade ammonium hydroxide. Ammonium formate may also be used as an eluent, at similar pH values. 8. The precise... in.) gives the best signal-to- noise ratio. Flow rate at which the postcolumn reagent is added should be reproducible between runs and must be the gradient pump flow; a pressure of 112–114 psi (eluent vessel must be able to withstand higher pressure) should achieve this flow rate with no significant increase in gradient pump pressure. Beware of air bubbles in the beaded mix- ing coil, which prevent... them completely. 16. Desalting may also be carried out by Bio-Gel P2 chromatography eluted in water. Acknowledgment The author wishes to thank Gail Evans for preparation of the manuscript and the UK Medical Research Council for funding. References 1. Hounsell, E. F. (1995) 1 H-NMR in the structural and conformational analysis of oligosac- charides and glycoconjugates, in Progress in NMR Spectroscopy,... investigated. 9. This flow rate is for a 100 × 4.6 mm Hypercarb S column. The smaller Glycosep H col- umn should not be run at flow rates in excess of 0.5 mL/min. 10. Great care should be taken to ensure thorough equilibration of the column prior to injecting samples because these columns are quite sensitive to changes in organic-phase composition. 11. The use of 0.1% TFA will cause quenching of fluorescence detection. 12. . by normal- or re-versed-phase high-performance liquid chromatography (HPLC) (8) or anion-exchange chro-matography, e.g., high-performance anion-exchange. HPAEC Analysis of O-Glycanase-Released Galβ 1-3 GalNAc1. Inject a 1- g aliquot of the O-Glycanase-released product, containing 320 ng of D-meli-biose as internal