HPLC Methods for the Fractionation and Analysis of Negatively Charged Oligosaccharides and Gangliosides

Adriana E. Manzi1, Bradley K. Hayes2

1 University of California San Diego, La Jolla, California, 2 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.21A
DOI:  10.1002/0471142727.mb1721as32
Online Posting Date:  May, 2001
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Abstract

This unit describes the fractionation and analysis of anionic oligosaccharides and gangliosides using anion‐exchange high‐performance liquid chromatography (HPLC). Saccharides or gangliosides are eluted in order of the number of negative charges they possess, although the charge‐to‐mass ratio can also contribute to elution position.

     
 
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Table of Contents

  • Basic Protocol 1: Fractionation of Released N‐Linked Oligosaccharides on DEAE HPLC Columns
  • Basic Protocol 2: Fractionation of Gangliosides on DEAE HPLC Columns
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Fractionation of Released N‐Linked Oligosaccharides on DEAE HPLC Columns

  Materials
  • HPLC‐grade or high‐quality deionized water (e.g., 18 MΩ Milli‐Q type)
  • recipe0.5 M sodium chloride (NaCl) in HPLC‐grade water (see recipe)
  • Oligosaccharide sample (Peptide N:glycosidase F–released and desalted; unit 17.13)
  • Sialylated oligosaccharide standards (e.g., Dionex or Oxford Glycosystems)
  • HPLC apparatus capable of two‐component gradient formation
  • DEAE HPLC column (TosoHaas TSK‐gel DEAE‐2SW, 25‐cm × 4.6‐mm i.d., 5‐mm particle size, or equivalent)
  • Additional reagents and equipment for desalting sample (units 17.13 & 17.14), and (if analyzing unlabeled sample) TBA assay (unit 17.18)

Basic Protocol 2: Fractionation of Gangliosides on DEAE HPLC Columns

  Materials
  • HPLC‐grade or high‐quality deionized (e.g., 18 MΩ Milli‐Q type) water
  • HPLC‐grade chloroform
  • HPLC‐grade methanol
  • recipe1:8:1 (v/v/v) chloroform/methanol/1 M aqueous ammonium acetate (see recipe)
  • Ganglioside sample (extracted and desalted; unit 17.3)
  • Ganglioside standards (G M1, G D1a and G T1b; e.g., Sigma, Boehringer Mannheim, or Calbiochem)
  • HPLC apparatus capable of two‐component gradient formation
  • DEAE HPLC column (TosoHaas TSK DEAE‐2SW, or comparable)
  • Additional reagents and equipment for high‐performance thin‐layer chromatography (HPTLC; if analyzing unlabeled sample unit 17.17) with chemical detection (unit 17.10) and for TBA assay (unit 17.18)
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Figures

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Literature Cited

   Baenziger, J.U. and Natowicz, M. 1981. Rapid separation of anionic oligosaccharide species by high performance liquid chromatography. Anal. Biochem. 112:357‐361.
   Green, E.D. and Baenziger, J.U. 1986 Separation of anionic oligosaccharides by high‐performance liquid chromatography. Anal. Biochem. 158:42‐49.
   Hallenbeck, P.C., Yu, F., and Troy, F.A. 1987. Rapid separation of polysialic acid by high‐performance liquid chromatography. Anal. Biochem. 161:181‐186.
   Hayes, B.K. and Varki, A. 1993a. Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N‐linked and O‐linked glycans labeled by UDP‐[6‐3H]N‐acetylgalactosamine. J. Biol. Chem. 268:16170‐16184.
   Manzi, A.E., Sjoberg, E.R., Diaz, S., and Varki, A. 1990. Biosynthesis and turnover of O‐acetyl and N‐acetyl groups in the gangliosides of human melanoma cells. J. Biol. Chem. 265:13091‐13103.
   Sjoberg, E.R., Manzi, A.E., Dell, A., Khoo, K.‐H., and Varki, A. 1992. Structural and immunological characterization of O‐acetyl GD2: Evidence that GD2 is an acceptor for ganglioside O‐acetyltransferase in human melanoma cells. J. Biol. Chem. 267:16200‐16211.
   Snyder, L.R., Glajch, J.L., and Kirkland, J.J. 1988. Practical HPLC Method Development. John Wiley & Sons, New York.
   Varki, A. and Kornfeld, S. 1983. The spectrum of anionic oligosaccharides released by endo‐β‐N‐acetylglucosaminidase H from glycoproteins. J. Biol. Chem. 258:2808‐2818.
Key References
   Hayes, B.K. and Varki, A. 1993b. Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N‐linked glycans labeled by UDP‐[6‐3H]galactose, CMP‐[9‐3H]N‐acetylneuraminic acid and [3H‐acetyl]AcCoA. J. Biol. Chem. 268:16155‐16169.
  Describes analysis of sialylated N‐linked oligosaccharides with and without prior sialidase treatment.
   Manzi et al., 1990. See above.
  Describes qualitative and semipreparative analysis of mono‐, di‐, and trisialylated gangliosides.
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