Protein Protocols Protein Protocols

842 Wong et al.
teins. The base is stabilized by Schiff base formation via reduction in sodium
cyanoborohydride. We have used this method, for example, to characterize the structure
and antigenicity of a series of reducing O-linked oligosaccharides released from
pigeon mucin (2).
2. Materials
1. Glycorelease N- and O-glycan recovery kit (Oxford GlycoSystems; Abingdon, UK).
2. Porous graphitized carbon (PGC) column (Hypersil Hypercarb, 100 mm × 4.6 mm, 7 µm,
Hypersil; Runcorn, UK).
3. Reagents used in reverse-phase (RP)-HPLC (see Subheading 3.2.): acetonitrile and
trifloroacetic acid (TFA) (Merck & Co.; Hoddesdon, UK).
4. Rat Thy-1 protein obtained by immunoaffinity purification and the lipid moiety removed
by phospholipase C (3).
5. Pigeon intestinal mucin obtained from the intestines of freshly killed pigeons by CsCl
density gradient (4).
6. Bovine fetuin, asialylated bovine fetuin, bovine serum albumin, chicken ovalbumin,
ExtraAvidin peroxidase conjugate, 4-chloro-1 naphthol, poly-L-lysine, ammonium carbonate,
and all reagents used in the modification and activation of NCM (see Subheading
3.4.): divinyl sulfone, dimethylformamide, sodium hydrogen carbonate, disodium carbonate,
ethylenediamine, glutaraldehyde, and methanol (Sigma; Poole, UK).
7. Bovine pancreatic RNase B (Oxford GlycoSystems; Abingdon, UK).
8. Immobilon-P nitrocellulose membranes (0.2-µm and 0.45-µm pore size) (Millipore Corp.;
Bedford, MA, USA).
9. Digoxigenin-labeled lectins of the glycan differentiation kit (Boehringer Mannheim;
Mannheim, Germany).
10. Biotinylated lectins (Vector Labs, Peterborough, UK).
11. Benchmark microplate reader (Bio-Rad Lab. Ltd.; Hemel Hempstead, UK).
3. Methods
All procedures are carried out at room temperature unless otherwise stated.
3.1. Hydrozinolysis for Release of N- and O-Linked Oligosaccharides
(e.g., O-Linked Glycoproteins) Using the Glycorelease Kit
1. Directly prior to hydrazinolysis, lyophilize glycoproteins overnight in a screw-capped)
vial and store in a desiccator in the presence of phosphorus pentoxide for 24 h.
2. Add 5 mL of anhydrous hydrazine to the vial, seal, and heat in a heating block at 60°C for
4 h for O-linked glycans (95°C for 5 h for N-linked glycans).
3. Cool the reaction vessel to room temperature and remove excess hydrazine by centrifugal
evaporation under reduced pressure (see Note 1).
4. Acetylate the released oligosaccharides by addition of 0.5 mL of ice-cold acetylation
buffer and 50 µL of acetylating reagent (supplied with the kit). Incubate mixture for
20 min at 4°C and then 30 min at room temperature.
5. Desalt oligosaccharides on a cation-exchange resin and lyophilize using a centrifugal
evaporator.
6. Dissolve dried samples in a minimal amount of pure water. Purify the released glycans
from peptide material by ascending paper chromatography using high-purity butanol–
ethanol–water (1641 by vol). Elute oligosaccharides at and a few centimeters in front