Protein Protocols Protein Protocols

828 Hounsell, Davies, and Smith
4. Elute the glycoprotein in 350 µL of H 2O.
5. Elute the salt with an additional 1 mL of H 2O.
3.2. Glycosidase Digestions
1. Dissolve 1 nmol of glycoprotein in 100 µL of H 2O, and boil for 30 min to denature. Remove
a 10% aliquot for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE), as a control for detection of enzyme digestion. Dry the remainder by lyophilization
(see Note 1).
2. Resuspend the glycoprotein in 500 µL of Endo H digestion buffer and 5 µL of toluene. Add
1 mU of Endo H/l nmol glycoprotein, and incubate at 37°C for 72 h (see Notes 2 and 3).
3. Precipitate the protein with a twofold excess of ice-cold ethanol and centrifuge at 15,000g
for 20 min. Wash the pellet three more times with ice-cold ethanol. Dry the protein pellet,
take up in water, and aliquot 10% (relative to original amount) for SDS-PAGE.
4. Dry the remaining pellet, and resuspend in neuraminidase/neuraminidase digestion buffer
at a concentration of 2 nmol of glycoprotein/10 µL of buffer. Incubate for 18 h at 37°C,
and then ethanol-precipitate and aliquot as in step 3.
5. Resuspend the remaining glycoprotein in 500 µL of PNGase F digestion buffer, 5 µL
toluene, and 1 U PNGase F/10 nmol glycoprotein. Incubate at 37°C for 72 h before precipitation
and aliquoting as in step 3 (see Note 4).
6. Digest the final pellet with O-glycosidase under the same conditions as for the PNGase F
digestion. Precipitate the pellet from ethanol washing and dry.
7. Apply all the pellet to SDS-PAGE.
8. The supernatants containing N- and O-linked oligosaccharides can be analyzed as discussed
in Chapters 111, 118, and 119.
4. Notes
1. The described procedure assumes approx 10% glycosylation of the glycoprotein. The
amount of glycoprotein treated may have to be increased to obtain oligosaccharides for
further analysis with less highly glycosylated glycoproteins.
2. PNGase F is stored at –20°C, and all other enzymes at 4°C. Endo H removes high-mannose
oligosaccharide chains, but not complex type.
3. The toluene is added to prevent bacterial growth.
4. The PNGase F digestion can be performed directly on the boiled glycoprotein if all N-linked
glycoprotein chains (both high mannose and complex) are required to be removed. Digests
may also be performed in 0.2 M sodium phosphate buffer, pH 8.4, but this will result in the
release of sialic acid residues as monosaccharides.
References
1. Yamamoto, K., Tsuji, T., and Osawa, T. (1993) Analysis of asparagine-linked oligosaccharides
by sequential lectin-affinity chromatography, in Methods in Molecular Biology,
vol. 14: Glycoprotein Analysis in Biomedicine (Hounsell, E. F., ed.), Humana, Totowa, NJ,
pp. 17–34.
2. Davies, M. J., Smith, K. D., and Hounsell, E. F. (1994) The release of oligosaccharides
from glycoproteins, in Methods in Molecular Biology, vol. 32: Basic Protein and Peptide
Protocols (Walker, J. M., ed.), Humana, Totowa, NJ, pp. 129–141.