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Monosaccharide Composition of Mucin 15915914Monosaccharide Composition of MucinsJean-Claude Michalski and Calliope Capon1. IntroductionMucin oligosaccharides are constructed by monosaccharide addition to form com-mon cores. This architecture limits the number of constituent monosaccharides.Monosaccharides commonly found in mucins may be divided into neutral (galactose[Gal]; fucose [Fuc]), hexosamines (N-acetylgalactosamine [GalNAc]; N-acetyl-glucosamine [GlcNAc]), and acidic compounds (sialic acids [NeuAc]). Additive het-erogeneity comes from the possible substitution with aglycone residues such as sulfate,phosphate, or acetate groups. Prior to their analysis, monosaccharides must be releasedfrom the oligosaccharide chain by acidic hydrolysis. Monosaccharide composition canalso be achieved on free oligosaccharide-alditols released from the native glycopro-tein by reductive alkaline treatment (β-elimination). In this case, GalNAc is convertedinto N-acetylgalactosaminitol (GalNAc-ol). Different methods are available for theanalysis of monosaccharides depending mainly on the amount of material available.Several techniques, such as gas-liquid chromatography (GLC) or high-performanceliquid chromatography (HPLC), allow both quantitative and qualitative analysis ofmonosaccharide mixtures. Other chromatographic or electrophoretic procedures aredescribed herein, but these only allow a rapid qualitative analysis of samples. Singleseparated monosaccharides may be further identified by physicochemical methodssuch as mass spectrometry (MS) or nuclear magnetic resonance.1.1. Release and Identification of Sialic AcidsSialic acids constitute a family of nine-carbon carboxylated sugars found in theexternal position on glycan chains. The diversity of sialic acids is generated by thepresence of various substituents present on carbon 4, 5, 7, 8, and 9. The substituent oncarbon 5 can be an amino, an acetamido, a glycolyl-amido, or a hydroxyl group anddefines the four major types of sialic acids: neuraminic acid (NeuAc), N-acetyl-neuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and keto-deoxy-nonulosonic acid (Kdn), respectively. Substituents of the hydroxyl groups present onFrom:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 160 Michalski and Caponcarbons 4, 7, 8, and 9 can be acetyl, lactyl, methyl, sulfate, or phosphate, anhydroforms can also occur (Fig. 1) (1,2). Most of the substituents, largely O-acetyl groupsare quite labile during acid or alkaline hydrolysis methods generally utilized for therelease of monosaccharides. Consequently, the study of sialic acid must be generallyconsidered independently of other monosaccharides. The study of sialic acid modifi-cations has been attempted after release and purification by improving the methods toavoid any destruction, and is achieved either with low concentrated acid solutions (3)or with enzymatic hydrolysis.Many techniques for detection and quantification of sialic acids have been described(1). These techniques differ widely in the initial purification of sialic acids from otherbiological contaminants. One of the most widely used assays is the detection of freeNeu5Ac and Neu5Gc acids by the thiobarbituric acid assay (TBA). Free sialic acidsreact with periodate under acidic conditions to produce β-formylpyruvic acid, whichcondenses with TBA to produce a purple chromogen (λmax= 549 nm). The assay issensitive to 1 nmol, but 2-deoxy-sugars interfere because they also condense withTBA to give a chromophore with a slightly lower λmax(532 nm). Powell and Hart (4)have introduced an HPLC adaptation of the periodate–TBA assay sensitive to 2 pmol,and requiring no prior purification of released sialic acids. The characterization ofreleased sialic acids can be achieved by chromatography: thin-layer chromatography,GLC (3), or HPLC (5–8). The last technique has higher sensitivity and resolvingpower. We have reported the HPLC separation of sialic acid quinoxalinones (8) thatallows the detection of sialic acids at the femtomole level.Fig. 1. The sialic acids. The nine-carbon backbone common to all known sialic acids may besubstituted by R1 or R2 substituents, giving a family of more than 30 different compounds. Monosaccharide Composition of Mucin 1611.2. Analysis of Monosaccharides by GLCGLC methods for identification of monosaccharides are powerful and extremely sen-sitive. Detection is usually by means of a flame ionization detector (FID), but sensitivitymay be increased by coupling the gas chromatograph to an MS instrument (electron orchemical impact). Prior to analysis, monosaccharides must be released by hydrolysis ofthe oligosaccharides or the glycoproteins and converted to a volatile derivative (9,10).1.3. Separation of Monosaccharides by HPLCHPLC has been widely used because of the advantages of allowing rapid and directquantification of underivatized or derivatized samples and the ability to characterizesamples through coelution with samples of known structures or through retention timecomparison. Separation methods are based on anion-exchange (11), size exclusion(12), ion suppression (13), reversed-phase (14), and, most recently, high-performanceanion-exchange chromatography (HPAEC).HPAEC takes advantage of the weakly acidic nature of carbohydrates to give highlyselective separations at high pH using strong anion-exchange pellicular resins (15). InHPAEC, strong alkaline solutions, usually NaOH, are used as eluent. Under these condi-tions, the hydroxyl groups of carbohydrates are converted to oxyanions with pKa values inthe range of 12–14. The anomeric hydroxyl group of the reducing sugar is more acidic thanthe others but each of the hydroxyl groups is characterized by a different pKa value (16);thus, the modification of some of the hydroxyl groups should greatly influence the elutionpositions (separation of anomeric and positional isomers) (17,18). Monosaccharidesreleased from glycoproteins by the previously mentioned hydrolysis methods can be rap-idly separated in less than 30 min. Because their molar responses are different, a calibra-tion curve must be established for each monosaccharide. When coupled with pulsedamperometric detection (PAD), HPAEC allows direct quantification of underivatizedmonosaccharides or carbohydrates at low picomole levels (10–50 pmol) with minimalsample preparation and purification. PAD utilizes a repeating sequence of three potentials.The most important potential is E1, the potential at which the carbohydrate oxidation cur-rent is measured. Potential E2 is a more positive potential that oxidizes the gold electrodeand completely removes the carbohydrate oxidation products. The third potential, E3,reduces the oxidized surface of the gold electrode in order to allow detection during the nextcycle at E1. The three potentials are applied for fixed periods referred to as t1, t2, and t3.1.4. Electrophoretic Separation of MonosaccharidesSince the early 1990s, capillary electrophoresis has become a good alternative andrapid procedure for analytical separation of microquantities of carbohydrate com-pounds including monosaccharides (19). Separation of native monosaccharides is gen-erally difficult owing to the lack of ionized groups and to their low extinctioncoefficients, which do not allow direct ultraviolet (UV) absorbance detection. Conse-quently, separation generally requires precolumn derivatization with reagents that con-tain a suitable chromophoric or fluorophoric group in order to facilitate separation andincrease the sensitivity of detection. As described under HPLC, the most commontagging methods are based on the reductive amination procedure, wherein the reduc-ing end of the sugar reacts with the primary amino group of the chromophore (20). 162 Michalski and CaponDifferent chromophores such as 2-aminopyridine (20), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) (21), ethyl-4-aminobenzoate, and 4-aminobenzonitrile (22),have been used for electrophoretic separation of monosaccharides.2. Materials2.1. Release and Identification of Sialic Acids1. 1000 mol wt cutoff dialysis tube (Bioblock Scientific, Illkirch, France).2. Dowex AG 50 W ì 8 (H+) (Bio-Rad, Hercules, CA).3. Dowex AG 3 ì 4A (HCOO) (Bio-Rad).4. Neuraminidase from Vibrio cholerae or Clostridium perfringens (Boehringer Mannheim,Indianapolis, IN).5. HPLC equipment with fluorescent detector.6. Lichrosorb RP 18 HPLC column (5-àm resin, 250 ì 4.6 mm) equipped with an RT30-4Lichrosorb RP18, 7-àm guard cartridge (Merck, Darmstadt, Germany).7. Stock solution: 2.35 mL of phosphoric acid (85%), and 28.1 g of sodium perchlorate in 1L of distilled water.8. Working solution: water:methanol:2X buffer stock (2:3:5).9. 1,2-Diamino-4,5-methylene dioxybenzene (DMB) (Merck).10. C18 column (250 ì 4.6 mm, particle size 5 àm) (Beckman, Fullerton, CA).11. DMBsialic acid HPLC solventsa. Solvent A: methanol:water (7:93 v/v).b. Solvent B: acetonitrile:methanol:water (11:7:82 v/v/v).2.2. Analysis of Monosaccharides by GLC1. Gas-liquid chromatograph fitted with an FID.2. Magnesium turnings (Acros Organics, Geel, Belgium).3. Sodium chloride and sulfuric acid (Sigma, St. Louis, MO).4. Meso-inositol (Sigma).5. Silver carbonate (Sigma).6. Acetic anhydride (Sigma).7. Heptane (Acros Organics, Sunnyvale, CA).8. Bis-silyltrifluoroacetamide (BSTFA) (Pierce, Austin, TX).9. Silicone OV 101 (BP1 phase, SGE).10. Sodium borohydride (Merck).11. Pyridine (Merck).12. Dichloromethane (Merck).13. Silicone BP 70 (SGE).14. Helium gas (Air Liquide, Paris, France).2.3. HPLC Separation Using Amino-Bonded Silica1. HPLC apparatus equipped with a gradient system.2. Refractive index detector.3. KromasilNH25 àm column (250 ì 4.6 mm) (Alltech, Deerfield, IL).2.4. Reversed-Phase HPLC of Pyridylamino-Monosaccharides1. Analytical ODS (C18) 5-àm HPLC column (4.6 ì 250 mm) (Zorbax, Interchim,Montluỗon, France).2. 0.25 M sodium citrate buffer, pH 4.0, containing 1% acetonitrile (Merck). Monosaccharide Composition of Mucin 1632.5. HPAEC-PADAll eluents and chemical products must be of the highest purity available.1. Gradient pump module (Dionex Bio-LC apparatus, Sunnyvale, CA).2. A model PAD-2 detector equipped with a gold working electrode. The following pulsepotentials and durations are used for detection: E1 = 0.05 V (t1= 360 ms); E2 = 0.70 V (t2= 120 ms); E3 = –0.50 V (t3 = 300 ms) The response time is set to 3 s.3. Eluent Degas module to sparge and pressurize the eluents with helium (Dionex).4. Postcolumn with a DQP-1 single-piston pump (Dionex).5. CarboPac PA-1 column (4 × 250 mm) (Dionex).6. CarboPac PA-1 guard (4 × 50 mm) (Dionex).7. CarboPac MA-1 column (4 × 250 mm) (Dionex).8. CarboPac MA-1 guard (4 × 50 mm) (Dionex).9. 18 M Ω deionized water (Milli-Q Plus System, Millipore, Bedford, MA).10. NaOH 50% solution with less than 0.1% sodium carbonate (Baker, Deventer, The Netherlands).11. Anhydrous sodium acetate (Merck).12. Acetic acid (glacial, HPLC grade; Merck).13. Eluents containing sodium acetate should be filtered through 0.45-µm nylon filters(Millipore) prior to use.14. Solvents for separation of neutral monosaccharides, hexosamines, and uronic acids (seeSubheading 3.3.3.2.).a. Eluent 1: Deionized water.b. Eluent 2: 25 mM NaOH and 0.25 mM sodium acetate.c. Eluent 3: 200 mM NaOH and 300 mM sodium acetate.d. Eluent 4: 125 mM NaOH and 10 mM sodium acetate.15. Solvents for HPAEC separation of sialic acids (see Subheading 3.3.3.3.).a. Eluent 1: Deionized water.b. Eluent 2: 5 mM NaOAc.c. Eluent 3: 5 mM acetic acid (glacial, HPLC grade; Merck).16. Solvents for separation of a mixture of unreduced and reduced monosaccharides (seeSubheading 3.3.3.4.).a. Eluent 1: Deionized water.b. Eluent 2: 1.0 M NaOH.17. Neu5Ac and Neu5Gc acid (Sigma).18. A mixture of sialic acids released from bovine submaxillary gland mucin (BSM) (Sigma)(see Subheding 3.1.1.).2.6. Electrophoretic Separation of Monosaccharides1. Capillary zone electrophoresis apparatus fitted with a UV detector (Beckman).2. Capillary tube (50 µm id × 65 cm) (Beckman). A part of the polyimine coating on thecapillary tube is removed by burning at a distance of 15 cm from the cathode, to allow UVdetection.3. 2-Aminoacridone (AMAC) (Lambda Fluoreszentechnologie GmbH, Graz, Austria) madeup to 0.1 M in acetic acid:dimethylsulfoxide (DMSO, Acros Organics, Sunnyvale, CA)(3:17 v/v). The solution is stored at –70°C.4. 1 M sodium cyanoborohydride (Merck) in water. This solution is made fresh for eachexperiment. 164 Michalski and Capon3. Methods3.1. Release and Identification of Sialic AcidsFigure 2 illustrates the separation of the different sialic acid species obtained afterhydrolysis of BSM. The different sialic acids may be characterized according to theirspecific retention times. Additionally, each sialic acid may be characterized by MSanalysis (8).3.1.1. Chemical Hydrolysis of Sialic Acids1. Suspend 1–10 mg of mucins in 5 mL of 2 M acetic acid in a Teflon-capped reaction tube.2. Hydrolyze for 5 h at 80°C.3. Dialyze the solution for 24 h against 20 vol of water (1000 mol wt cutoff tubing).4. Lyophilize the diffusate. Direct analysis can be made at this stage.5. Further purify sialic acids as follows:a. Redissolve the dialysate in 1 mL of water.b. Load the sample on a Dowex AG 50W × 8 (H+) (Bio-Rad) column (10 mL).c. Wash the column with 100 mL of water.d. Lyophilize the effluent.e. Resuspend the lyophilysate in 1 mL of water.Fig. 2. HPLC separation of sialic acid quinoxalinones obtained after mild acid hydrolysis ofBSM. Ac, acetyl; Lt, lactyl; Gc, glycolyl. Monosaccharide Composition of Mucin 165f. Load the sample on a Dowex AG 3 × 4A (HCOO–) (Bio-Rad) column (1 mL).g. Wash the column successively with 7 mL of 10 mM , 7 mL of 1 M and 7 mL of 5 Mformic acid.h. Pool the fractions and lyophilize.3.1.2. Enzymatic Release of Sialic Acid1. Resuspend 1–10 mg of mucin in 2 mL of 100 mM HEPES-KOH, pH 7.0, 150 mM NaCl,0.5 mM MgCl2, and 0.1 mM CaCl2.2. Add 200 mU/mL of V. cholerae or 40 mU/mL C. perfringens enzyme.3. Introduce the solution in a dialysis tube (1000 mol wt cutoff) and dialyze against 5 mL ofthe same solvent at 37°C overnight.4. Collect the filtrate and purify the sialic acid as in Subheading 3.1.1.3.1.3. TBA-HPLC Quantification of Sialic Acids3.1.3.1. TBA REACTION1. Sialic acids are released from mucins by mild acid hydrolysis as described in Subhead-ing 3.1.1. The TBA assay is performed essentially according to Warren (23).2. Place 40 µL of free sialic acid solution (10–100 pmol/100 µL in water) in an Eppendorf tube.3. Add 20 µL of sodium periodate (128 mg of sodium metaperiodate, 1.7 mL of phosphoricacid, and 1.3 mL of water).4. After 20 min at room temperature, add slowly 0.1 mL of 10% sodium arsenite in 0.1 NH2SO4,and 0.5 M Na2SO4.5. When the solution appears yellow-brown, gently vortex the tubes.6. Add 0.6 mL of 0.6% TBA (0.6 g of TBA [Sigma] in 0.5 M Na2SO4[Merck]).7. After mixing, cap the tubes and heat at 100°C for 15 min.8. Chill the tubes on ice and centrifuge before HPLC analysis.3.1.3.2. HPLC ANALYSIS (FIG. 3)1. Equilibrate the column in the working solution.2. Elute in the isocratic mode at a flow rate of 1 mL/min.3. Run UV detection at 549 nm.4. Quantify the sialic acid by integrating the surface of the sialic acid. Obtain the chromophorepeak calibration curve with pure sialic acid solution (1–5 µg of sialic acid in 40 µL of water).5. Wash the column extensively with 50% acetonitrile in water after use.3.1.4. Characterization and Quantification of Sialic Acids by HPLC3.1.4.1. DERIVATIZATION WITH DMB1. Heat sialic acid samples released by mild hydrolysis in 7 mM DMB, 0.75 M β-mercaptoethanol, and 18 mM sodium hydrosulfite in 1.4 M acetic acid (100–200 µL) for2.5 h in the dark.2. Inject 10 µL of the reaction mixture on the C18 column.3.1.4.2. ELUTION BY HPLC1. Equilibrate the column in 65% solvent A–35% solvent B.2. Elute using a linear gradient from 65% A/35% B to 100% B over 60 min followed byisocratic elution by 100% B for 10 min at a flow rate of 1 mL/min.3. Achieve on-line fluorescent detection at an emission wavelength of 448 nm and excita-tion wavelength of 373 nm with a response time of 0.5 s. 166 Michalski and Capon3.2. Analysis of Monosaccharides by GLCComplete hydrolysis of oligosaccharide chains may be obtained using concentratedacid solutions.3.2.1. Trifluoroacetic Hydrolysis1. Dissolve the oligosaccharide-alditol sample or the native glycoprotein in 0.5 mL of a 4 Msolution of trifluoroacetic acid (TFA) or a mixture of formic acid:water:TFA (3:2:1 v/v/v).2. Heat at 100°C for 4 h in Teflon-capped tubes.3. After hydrolysis, remove the acid by repeated evaporation under reduced pressure. Evapo-ration is completed by the addition of ethanol.3.2.2. Formic Acid–Sulfuric Acid Hydrolysis1. Dissolve oligosaccharide-alditols or native glycoproteins in 0.5 mL of 50% aqueous for-mic acid and hydrolyze for 5 h at 100°C in a Teflon-capped tube.2. Repeat step 1 using 0.25 M aqueous sulfuric acid for 18 h at 100°C.3. Neutralize the hydrolysate with barium carbonate powder, filter, and concentrate to dry-ness (see Note 1).3.2.3. MethanolysisMethanolysis is a widely used method for hydrolysis of both oligosaccharides andnative glycoproteins.Fig. 3. HPLC analysis of TBA chromophores. 2, NeuAc chromophore; 3, deoxyhexose chro-mophore. Monosaccharide Composition of Mucin 1673.2.3.1. PREPARATION OF METHANOL–HCL REAGENT1. Obtain anhydrous methanol by refluxing with magnesium turnings (1 h) followed by dis-tillation in a dry all-glass apparatus.2. Generate gaseous HCl by slow addition (10–20 drops/min) of sulfuric acid to 250 g ofsolid NaCl.3. Dry the hydrogen chloride gas through moisture traps containing concentrated sulfuric acid.4. Then bubble hydrogen chloride gas through anhydrous methanol for 3 to 4 h.5. Standardize the methanol–HCl reagent to 0.5 M by titration with NaOH and dilution withanhydrous methanol (see Note 2).3.2.3.2. METHANOLYSIS OF OLIGOSACCHARIDE OR MUCUS GLYCOPROTEIN(24)1. Freeze-dry carefully in Teflon-capped tubes (complete dehydration of samples is themain condition of success) amounts of glycoproteins or oligosaccharides correspondingto 10 µg of total sugar to which 1 µg of mesoinositol is added as an internal standard.2. Add 0.5 mL of methanol–HCl reagent.3. Heat at 80°C for 24 h.3.2.4. GLC Analysis of Monosaccharides as Their Methylglycoside (25)Trimethylsilylated DerivativesGLC analysis allows the determination of the monosaccharide composition of gly-cans with amounts of total sugars not exceeding 1 µg (15–20 pmol of glycoproteins). Itconsists of the methanolysis of previously purified glycoprotein, followed by a re-N-acetylation and a trimethylsilylation leading to trimethylsilylation-derivatives.3.2.4.1. METHANOLYSIS AND DERIVATIZATION1. Mix amounts of purified glycoproteins corresponding to 0.5 µg of total sugars with 200 µLof 0.5 M methanol-HCl mixture for 24 h at 80°C.2. After cooling the tube, neutralize the acidic solution by adding silver carbonate to give apH of 6.0-7.0 as controlled with pH paper.3. Re-N-acetylate by adding 10 µL of acetic anhydride and keep overnight at room tem-perature.4. Centrifuge at 2000g for 5 min and collect the supernatant.5. To eliminate fatty acid methyl esters, wash the methanolic phase two times with 200 µLof heptane (remove the upper phase).6. Dry the methanolic lower phase under a stream of nitrogen.7. Trimethylsilylate with 20 µL of BSTFA in the presence of 10 µL of pyridine for 1 h atroom temperature.8. Apply 1–5 µL of the solution of trimethylsilylated methylglycosides to GLC.3.2.4.2. GAS CHROMATOGRAPHY CONDITIONSA typical GLC chromatogram of TMS derivatives is given in Fig. 4. Neutral monosac-charides generally provide several peaks corresponding to pyrano, furano, α, and β forms.1. Use a FID gas chromatograph and a glass solid injector (moving needle).2. Use a capillary column (25 m × 0.33 mm) of silicone OV 101.3. Use carrier gas helium at a pressure of 0.5 bar.4. Program the oven temperature from 120 to 240°C at 2°C/min.5. Use injector and detector temperatures of 240 and 250°C, respectively. 168Michalski and Capon168Fig. 4. GLC separation of trimethylsilylated deriviatives of monosaccharides released by methanolysis from β-eliminated intes-tinal mucin oligosaccharide-alditols. [...]... acid and check the resolution. The total running time is below 30 min. 4. Load the sample and start the run. 5. Apply a two-step run at a flow rate of 1.0 mL/min (see Table 3). Fig. 7. HPLC separation of pyridylamino derivatives of monosaccharides by reversed-phase HPLC Ultrasphere ODS (4.6 × 250 mm). 1, PA-Gal; 2, PA-Glc; 3, PA-Man; 4, PA-Rib; 5, PA- Fuc; 6, PA-Rham; 7, PA-ManNAc; 8, PA-deoxy-Rib;... J. P., Breckenridge, W. C., and Vincendon, G. (1972) Analysis of monosaccha- rides by gas-liquid chromatography of the O-methyl glycosides as trifluoroacetate deriva- tives. Application to glycoproteins and glycolipids. J. Chromatogr. 69, 291–304. 25. Chaplin, M. F. (1982) A rapid and sensitive method for the analysis of carbohydrate com- ponents in glycoproteins using gas-liquid chromatography. Anal.... (1989) High performance liquid chromatography of reducing carbohydrates as strongly ultraviolet ab- sorbing and electrochemically sensitive 1-phenyl-3-methyl-5-pyrazolone derivatives. Anal. Biochem. 180, 351–357. 42. Fu, D. and O’Neill, R. A (1995) Monosaccharide composition analysis of oligosaccha- rides and glycoproteins by high performance liquid chromatography. Anal. Biochem. 227, 377–384. 43. Alpenfelsm... Achieve on-line fluorescent detection at an emission wavelength of 448 nm and excita- tion wavelength of 373 nm with a response time of 0.5 s. 180 Michalski and Capon 33. Jentoft, N. (1985) Analysis of sugars in glycoproteins by high-pressure liquid chromatog- raphy. Anal. Biochem. 148, 424–433. 34. Gisch, D. J. and Pearson, J. D. (1988) Determination of monosaccharides in glycoproteins by reverse-phase... reverse-phase high perfor- mance liquid chromatography in the low and subpicomole range. Anal. Biochem. 195, 160–167. 40. Lin, J. K. and Wu, S. S. (1987) Synthesis of dansyl hydrazine and its use in the chromato- graphic determination of monosaccharides by thin-layer and high performance liquid chro- matography. Anal. Biochem. 591, 1320–1326. 41. Honda, S., Akao, E., Suzuki, S., Okada, M., Kakahi, K., and. .. J., and Stone, B. A. (1983) Detection of neutral and amino sugars form glycoproteins and polysaccharides as their alditol-acetates. J. Chromatogr. 256, 419–427. 27. Takemoto, H., Hase, S., and Ikenaka ,T. (1985) Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino-derivatives by high performance liquid chro- matography. Anal. Biochem. 145, 245–250. 28. Manzi, A. E. and. .. monosaccharides in glycoproteins by re- verse-phase by high performance liquid chromatography. Anal. Biochem. 215, 243–252. 47. Hernandez, L. M., Ballon, L., Alvarado, E., Gillea-Castro, B. L., Burlingame, A. L., and Ballon, C. E. (1989) A new Saccharomyces cerevisiea mnn Mutant N-linked oligosaccha- ride structure. J. Biol. Chem. 264, 11,849–11,856. 48. Hemmrich, S., Bertozzi, C. R., Leffler, H., and Rosen,... (33,34) p-Bromo-benzoyl; UV 250 nm 1 ng Silica; dichloromethane:ethyl acetate (25:4) (35) naphtoyl-benzoyl UV260–254 nm 300 ng Silica; ethyl acetate:hexane (1:5) (36) Dns-hydrazide Fluorescence 2–5 pmol C18 (37) DABS hydrazide Fluorescence 2–5 pmol C18 (80% acetone:0.08 M CH 3 COOH) (37,38) Fmoc hydrazide Fluorescence 1 pmol C18 (CH 3 CN:AcCOOH) (39) Dansyl-hydrazide 425 nm 10 pmol C18 (40) 1-phenyl-3-methyl-5-pyrazolone... (w/v) polyacrylamide gels containing 0.67 % (w/v) N-N'-meth- ylene-bisacrylamide. The final concentrations of N,N,N',N'-tetramethylenediamine and ammonium persulfate are 0.1% (v/v) and 0.1% (w/v), respectively. The gel dimensions are 140 mm id by 0.5 mm thick. Electrophorese samples are initially at 100 V for 30 min, then at 200 V for 30 min, and finally at 500 V for 90 min. 7. Use bromophenol... J. and Chiesa, C. (1994) Capillary electrophoresis of carbohydrates. Glyco- biology 4, 397–412. 20. Honda, S., Iwase, S., Makino, A., and Fujiwara, S. (1989) Simultaneous determination of reducing monosaccharides by capillary zone electrophoresis as the borate complexes of N-2-pyridyl-glycamines. Anal. Biochem. 176, 72–77. 21. Chiesa, C. and Horvath, C. (1993) Capillary zone electrophoresis of malto-oligosaccharides derivatized . reversed-phaseHPLC Ultrasphere ODS (4.6 × 250 mm). 1, PA-Gal; 2, PA-Glc; 3, PA-Man; 4, PA-Rib; 5, PA-Fuc; 6, PA-Rham; 7, PA-ManNAc; 8, PA-deoxy-Rib; 9, PA-GlcNAz;. chromophores such as 2-aminopyridine (20), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) (21), ethyl-4-aminobenzoate, and 4-aminobenzonitrile (22),have been used