Fractionation and Analysis of Neutral Oligosaccharides by HPLC

Bradley K. Hayes1, Ajit Varki2

1 University of Alabama at Birmingham, Birmingham, Alabama, 2 University of California Sun Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.21B
DOI:  10.1002/0471142727.mb1721bs32
Online Posting Date:  May, 2001
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Abstract

This unit describes the fractionation and analysis of neutral oligosaccharides by high‐performance liquid chromatography (HPLC) on bonded amine columns. Separation is based upon hydrogen bonding between the NH2 groups of the column and the hydroxyl groups of the oligosaccharides. A support protocol describes the reduction and desalting of neutral oligosaccharides with sodium borohydride.

     
 
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Table of Contents

  • Basic Protocol 1: Separation of Neutral Oligosaccharides on a Bonded Amine HPLC Column
  • Support Protocol 1: Sodium Borohydride Reduction and Desalting of Neutral Oligosaccharides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Separation of Neutral Oligosaccharides on a Bonded Amine HPLC Column

  Materials
  • Oligosaccharide sample
  • Oligosaccharide standards (e.g., Dionex or Oxford Glycosystems)
  • HPLC‐grade water
  • HPLC‐grade acetonitrile
  • HPLC apparatus capable of two‐component gradient formation
  • Bonded amine HPLC column (e.g., Varian Micropak AX‐5 or Rainin LC‐NH 2)
  • Additional reagents and equipment for exoglycosidase digestion (optional; units 17.1317.13 & 17.18), and reduction and desalting of neutral oligosaccharides (see 17.21)

Support Protocol 1: Sodium Borohydride Reduction and Desalting of Neutral Oligosaccharides

  • recipe1 M sodium borohydride (NaBH 4) in 0.2 M sodium borate (Na 2B 4O 7; see recipe)
  • 5% acetic acid/95% methanol
  • Mixed‐bed anion and cation resin (Amberlite MB‐3 or equivalent)
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Figures

Videos

Literature Cited

   Blanken, W.M., Bergh, M.L.E., Koppen, P.L., and van den Eijnden, D.H. 1985. High pressure liquid chromatography of neutral oligosaccharides: Effects of structural parameters. Anal. Biochem. 145:322‐330.
   Brockhausen, I., Narasimhan, S., and Schachter, H. 1988. The biosynthesis of highly branched N‐glycans: Studies on the sequential pathway and functional role of N‐acetylglucosaminyltransferases I, II, III, IV, V and VI. Biochimie 70:1521‐1533.
   Koenderman, A.H.L., Koppen, P.L., Koeleman, A.M., and van den Eijnden, D.H. 1989. N‐acetylglucosaminyltransferases III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct. Eur. J. Biochem. 181:651‐655.
   Mellis, S.J. and Baenziger, J.U. 1981. Separation of neutral oligosaccharides by high pressure liquid chromatography. Anal. Biochem. 114:276‐280.
   Mellis, S.J. and Baenziger, J.U. 1983. Size fractionation of anionic oligosaccharides and glycopeptides by high‐pressure liquid chromatography. Anal. Biochem. 134:442‐449.
   Sampath, D., Varki, A., and Freeze, H.H. 1992. The spectrum of incomplete N‐linked oligosaccharides synthesized by endothelial cells in the presence of Brefeldin A. J. Biol. Chem. 279:4440‐4455.
   Schacter, H., Brockhausen, I., and Hull, E. 1989. High‐performance liquid chromatography assays of N‐acetylglucosaminyltransferases involved in N‐ and O‐linked synthesis. Methods Enzymol. 179:351‐396.
   Snyder, L.R., Glajch, J.L., and Kirkland, J.J. 1988. Practical HPLC Method Development. John Wiley & Sons, New York.
   Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. 1988. Analysis of N‐linked oligosaccharides using a two‐dimensional mapping technique. Anal. Biochem. 171:73‐90.
   Tomiya, N., Yuan, Y.C., Yoshida, T., Wada, Y., Away, J., Kurono, M., and Takahashi, N. 1991. Calculated two‐dimensional sugar map of pyridylaminated oligosaccharides: Elucidation of the Jack Bean α‐mannosidase digestion pathway of Man9GlcNAc2. Anal. Biochem. 193:90‐100.
Key References
   Blanken et al., 1985. See above.
  Describes important parameters for optimal resolution of neutral oligosaccharides.
   Mellis and Baenziger, 1981. See above.
  Describes important parameters for optimal resolution of neutral oligosaccharides.
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