A general approach to desalting oligosaccharides released from glycoproteins.
Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and beta-eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.
Packer
N H
NH
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia. nicolle.packer@mq.edu.au
Lawson
M A
MA
Jardine
D R
DR
Redmond
J W
JW
D000581:Amidohydrolases / Q000378:metabolism; D001894:Borohydrides; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006108:Graphite; D006801:Humans; D006834:Hydrazines; D016464:Lysosomal Storage Diseases / Q000652:urine; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification* / Q000652:urine; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012492:Salts; D012972:Sodium Hydroxide
D001894:Borohydrides; D006023:Glycoproteins; D006834:Hydrazines; D009844:Oligosaccharides; D012492:Salts; C029424:hydrazine; D012972:Sodium Hydroxide; D006108:Graphite; C025364:sodium borohydride; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase