Total Compositional Analysis by High‐Performance Liquid Chromatography or Gas‐Liquid Chromatography

Adriana E. Manzi1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.19A
DOI:  10.1002/0471142727.mb1719as32
Online Posting Date:  May, 2001
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Abstract

Once the presence of carbohydrate in a glycoprotein has been confirmed, the next step is to determine the precise molar ratio of its monosaccharide constituents. The analysis involves two major phases. The release of the individual monosaccharides is achieved by methanolysis (described here), total acid hydrolysis, or enzymatic release of sialic acids. The resulting mixtures of monosaccharides are then analyzed by methods described in this unit: fractionation, characterization, and quantitation by high‐performance liquid chromatography using anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) and other HPLC systems or by gas‐liquid chromatography with flame ionization detection. The identity of the individual monosaccharides is determined by comparison with known standards processed and analyzed in the same way.

     
 
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Table of Contents

  • Strategic Planning
  • Compositional Analysis of Free Monosaccharides by HPAEC‐PAD
  • Basic Protocol 1: Analysis of Mixtures of Neutral Monosaccharides and Hexosamines
  • Basic Protocol 2: Analysis of Mixtures of Neutral Monosaccharides, Hexosamines, and Uronic Acids
  • Basic Protocol 3: Analysis of Mixtures of Sialic Acids
  • Alternate Protocol 1: Compositional Analysis of Free Sialic Acids by Amine‐ Adsorption Ion‐Suppression HPLC with UV Detection
  • Basic Protocol 4: Compositional Analysis by GLC‐FID
  • Support Protocol 1: Quantitative Release of Neutral Monosaccharides, Hexosamines, and Uronic Acids by Methanolysis
  • Support Protocol 2: Preparation of Volatile Derivatives of Free Glycoses
  • Support Protocol 3: Preparation of Volatile Derivatives of Methyl Glycosides
  • Support Protocol 4: Preparation of Monosaccharide Standard Solutions
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Mixtures of Neutral Monosaccharides and Hexosamines

  Materials
  • Milli‐Q‐purified water: water deionized by passage through a five‐stage Milli‐Q Plus system (Millipore)
  • Certified 50% (19.3 M) sodium hydroxide containing <0.1% sodium carbonate (Fisher)
  • Nitrogen
  • Monosaccharide standard mixture (see protocol 9)
  • Monosaccharide sample prepared by acid hydrolysis (unit 17.16: protocol 3, protocol 1 when analyzing only fucose)
  • Filtering unit with 0.45‐µm Nylon 66 membranes (Alltech)
  • High‐pH anion‐exchange chromatography system with pulsed amperometric detector consisting of gradient pump, eluant degassing module, and PAD cell equipped with thin gasket (Dionex)
  • CarboPac PA‐1 column (250 × 4 mm; Dionex)
  • CarboPac PA Guard column (3 × 25 mm; Dionex)

Basic Protocol 2: Analysis of Mixtures of Neutral Monosaccharides, Hexosamines, and Uronic Acids

  • Sodium acetate (CH 3CO 2Na⋅3H 2O, FW 136.08; ACS certified grade)

Basic Protocol 3: Analysis of Mixtures of Sialic Acids

  • Glacial acetic acid (17.4 M)
  • N‐acetylneuraminic acid (Neu5Ac, MW 309.3; Boehringer Mannheim)
  • N‐glycolylneuraminic acid (Neu5Gc, MW 325.3; Sigma)
  • Standard mixture: sialic acids prepared from bovine submaxillary mucin (BSM) using the procedures in unit 17.16 ( protocol 2Basic Protocol 2 or protocol 3Alternate Protocol 1) or unit 17.12, or equimolar mixture of Neu5Gc and Neu5Ac processed as for the sample

Alternate Protocol 1: Compositional Analysis of Free Sialic Acids by Amine‐ Adsorption Ion‐Suppression HPLC with UV Detection

  Materials
  • Monobasic sodium phosphate (anhydrous, MW 120.0; reagent grade, Sigma)
  • Milli‐Q‐purified water: water deionized by passage through five‐stage Milli‐Q Plus system (Millipore)
  • Acetonitrile (HPLC grade, Fisher)
  • N‐acetylneuraminic acid (Neu5Ac, MW 309.3; Boehringer Mannheim)
  • N‐glycolylneuraminic acid (Neu5Gc, MW 325.3; Sigma)
  • Standard mixture: sialic acids from bovine submaxillary mucin (BSM) prepared using procedures in unit 17.16 or unit 17.12, or equimolar mixture of Neu5Ac and Neu5Gc processed as for the sample
  • Sialic acid sample prepared by acid hydrolysis (unit 17.16, protocol 2Basic Protocol 2 or protocol 3Alternate Protocol 1) or enzymatic release (unit 17.12)
  • HPLC apparatus with pump and UV detector
  • Filtering unit with 0.45‐µm Nylon 66 membranes (Alltech)
  • Micropak AX‐5 (300 mm × 7.8 mm i.d., particle size 9 µm; Varian)
  • Micropak AX‐5 Guard column (4 cm × 4 mm)
  • Additional reagents and equipment for de‐O‐acetylation (unit 17.18)

Basic Protocol 4: Compositional Analysis by GLC‐FID

  Materials
  • Monosaccharide standard mixture (see protocol 8) prepared in the same manner as the sample
  • Monosaccharide sample prepared by acid hydrolysis (unit 17.16, protocol 3) followed by derivatization by peracetylation (see protocol 7) and/or by methanolysis ( protocol 6) followed by derivatization by pertrimethylsilylation (see protocol 8)
  • Internal standard prepared in the same manner as the sample
  • 0.25 mm × 30 m 5% DB‐5 fused silica capillary column (J&W Scientific)
  • Gas chromatograph with dual‐flame ionization detector
  • Helium (research grade)
  • Hydrogen and air lines (for FID detector only)

Support Protocol 1: Quantitative Release of Neutral Monosaccharides, Hexosamines, and Uronic Acids by Methanolysis

  Materials
  • Glycoconjugate sample containing 5 to 50 µg total sugar
  • Internal monosaccharide standard: monosaccharide that does not occur naturally in the sample (e.g., monosaccharide alditol that yields only one peak upon gas chromatography, see unit 17.16; see protocol 9 for monosaccharide preparation)
  • Monosaccharide standards: monosaccharides expected to be found in the sample (see protocol 9 for preparation)
  • Phosphorus pentoxide (Fisher)
  • Acetyl chloride (FW 78.50, 98% pure; Aldrich)
  • Methanol (anhydrous, 99% pure, Aldrich; store in desiccator)
  • Pyridine (anhydrous silylation grade, Pierce; store in desiccator)
  • Acetic anhydride
  • 1.5‐ml glass Reacti‐Vials (Pierce) with Teflon‐lined screw caps, either new (rinsed with water, then with ethanol, and dried) or recipeacid‐cleaned (see recipe)
  • Heating block or oven
  • Nitrogen or vacuum evaporation system (Speedvac or shaker‐evaporator)

Support Protocol 2: Preparation of Volatile Derivatives of Free Glycoses

  Materials
  • 10 mg/ml sodium borohydride in 1 M ammonium hydroxide (ACS reagent grade)
  • Monosaccharide sample prepared by acid hydrolysis (unit 17.16, protocol 4Basic Protocol 3, or protocol 1Basic Protocol 1 when analyzing only fucose)
  • Neutral sugar standard mixture (see protocol 9) submitted to acid hydrolysis (unit 17.16, protocol 4Basic Protocol 3)
  • Sodium borohydride (FW 37.8; store in desiccator)
  • Glacial acetic acid (FW 60.05; 99.99% pure)
  • 1% acetic acid in methanol (anhydrous, 99% pure)
  • Acetic anhydride (99% pure)
  • Toluene
  • Chloroform (anhydrous, 99% pure)
  • Acetone (99.9% pure, HPLC grade)
  • Phosphorous pentoxide (as desiccant)
  • 3.5‐ml glass vials with Teflon‐lined screw caps
  • Nitrogen evaporation unit (Reacti‐Vap Evaporator, Reacti‐Therm Heating Module, and Reacti‐Block B‐1 or S‐1, Pierce)
  • Heating block or oven, 100°C
  • Tabletop centrifuge
  • Reacti‐Vials (Pierce) or other glass test tubes

Support Protocol 3: Preparation of Volatile Derivatives of Methyl Glycosides

  Materials
  • Dried methanolyzed samples and standards (see protocol 6, step )
  • Tri‐Sil Reagent [2:1:10 (v/v/v) hexamethyldisilazane/trimethylchlorosilane/ pyridine; Pierce]
  • 96% hexane (HPLC grade)
  • Nitrogen stream
  • Tabletop centrifuge
  • Reacti‐Vials (Pierce)

Support Protocol 4: Preparation of Monosaccharide Standard Solutions

  Materials
  • NaOH pellets
  • Monosaccharide standards
  • Milli‐Q‐purified water: water deionized by passage through a Milli‐Q Plus system (Millipore)
  • Vacuum desiccator
  • Glass vials
  • 100‐ml volumetric flasks
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Figures

Videos

Literature Cited

Literature Cited
   Albersheim, P., Nevins, D.J., English, P.D., and Karr, A. 1967. A method for the analysis of sugars in plant cell‐wall polysaccharides by gas‐liquid chromatography. Carbohydr. Res. 5:340‐345.
   Bergh, M.L., Koppen, P., and Van den Eijnden, D.H. 1981. High‐performance liquid chromatography of sialic acid–containing oligosaccharides. Carbohydr. Res. 94:225‐229.
   Clarke, A.J., Sarabia, V.S., Keenleyside, W., MacLachlan, P.R., and Whitfield, C. 1991. The compositional analysis of bacterial extracellular polysaccharides by high‐performance–anion‐exchange chromatography. Anal. Biochem. 199:68‐74.
   Diaz, S. and Varki, A. 1985. Metabollic labeling of sialic acids in tissue culture cell lines: Methods to identify substituted and modified radioactive neuraminic acids. Anal. Biochem. 150:32‐46.
   Hardy, M.R. and Townsend, R.R. 1988. Separation of positional isomers of oligosaccharides and glycopeptides by high‐performance anion‐exchange chromatography with pulsed amperometric detection. Proc. Natl. Acad. Sci. U.S.A. 85:3289‐3293.
   Hardy, M.R. and Townsend, R.R. 1989. Separation of fucosylated oligosaccharides using high pH anion‐exchange chromatography with pulsed amperometric detection. Carbohydr. Res. 188:1‐7.
   Hardy, M.R. and Townsend, R.R. 1994. High‐pH anion exchange chromatography of glycoprotein‐derived carbohydrates. Methods Enzymol. 230:208‐225.
   Kamerling, J.P. and Vliegenthart, J.F.G. 1989. In Clinical Biochemistry, Vol. 1: Principles, Methods, Applications. Mass Spectrometry. (A.M. Lawson, ed.) pp. 175‐263. Walter de Gruyter, Berlin.
   LaCourse, W.R. and Johnson, D.C. 1991. Optimization of waveforms for pulsed amperometric detection (p.a.d.) of carbohydrates following separation by liquid chromatography. Carbohydr. Res. 215:159‐178.
   Lee, Y.C. 1990. High‐performance ion‐exchange chromatography for carbohydrate analysis. Anal. Biochem. 189:151‐162.
   Manzi, A.E., Diaz, S., and Varki, A. 1990. High‐pressure liquid chromatography of sialic acids on a pellicular resin anion‐exchange column with pulsed amperometric detection: A comparison with six other systems. Anal. Biochem. 188:20‐32.
   Mellis, S.J. and Baenziger, J.U. 1981. Separation of neutral oligosaccharides by high‐performance liquid chromatography. Anal. Biochem. 114:276‐280.
   Mellis, S.J. and Baenziger, J.U. 1983. Size fractionation of anionic oligosaccharides and glycopeptides by high‐performance liquid chromatography. Anal. Biochem. 134:442‐449.
   Reddy, G.P. and Bush, C.A. 1991. High‐performance anion‐exchange chromatography of neutral milk oligosaccharides and oligosaccharide alditols derived from mucin‐type glycoproteins. Anal. Biochem. 198:278‐284.
   Reinhold, V.N., 1972. Gas‐liquid chromatographic analysis of constituents carbohydrates in glycoproteins. Methods Enzymol. (Part B) 25:244‐260.
   Rickert, S.J. and Sweeley, C.C. 1978. Quantitative analysis of carbohydrate residues of glycoproteins and glycolipids by gas‐liquid chromatography. An appraisal of experimental details. J. Chromatogr. 147:317‐326.
   Schauer, R. 1978. Characterization of sialic acids. Methods Enzymol. 50:64‐89.
   Schauer, R. and Corfield, A.P. 1982. In Sialic Acids: Chemistry, Metabolism and Function (R. Schauer, ed.) pp. 51‐57. Springer‐Verlag, New York.
   Van Riel, J. and Olieman, C. 1991. Selectivity control in the anion‐exchange chromatographic separation of saccharides in dairy products using pulsed amperometric detection. Carbohydr. Res. 215:39‐46.
   Wong, C.G., Sung, S.‐S., J., and Sweeley, C.C. 1980. Analysis and structural characterization of amino sugars by gas‐liquid chromatography and mass spectrometry. Methods Carbohyd. Chem. 8:5565.
   York, W.S., Darvill, A.G., McNeil, M., Stevenson, T.T., and Albersheim, P. 1985. Isolation and characterization of plant cell wall and cell wall components. Methods Enzymol. 118:3‐54.
Key References
   Kamerling and Vliegenthart, 1989. See above.
  Descriptions of the principles of each method with examples of their application.
   Hardy and Townsend, 1994. See above.
   Manzi et al., 1990. See above.
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