Protein Protocols Protein Protocols

Carbohydrate Electrophoresis 859
Fig. 2. Profiles of O-linked oligosaccharides from several glycoproteins. ANTS-labeled Ser/
Thr-lined oligosaccharides released using hydrazine at 60°C from four different glycoproteins
are shown. Lane 1 contains an oligosaccharide ladder standard of partially hydrolyzed wheat
starch with G4 representing glucotetraose; lane 2, porcine stomach mucin, Type II; lane 3,
bovine submaxillary mucin, Type I; lane 4, bovine fetuin; and lane 5, human chorionic gonadotropin
(hCG). Lane 6 contains the NeuAc(α2-3)Gal(β1-3)[NeuAc(α2-6)]GalNAc and
Gal(β1-3)GalNAc standards. The images presented in this review were obtained by imaging
the gels following electrophoresis using a CCD-based imaging system (Glyko).
protein, the amount of glycosylation, and the degree of oligosaccharide heterogeneity. In
general, the amount of glycoprotein required increases with the size of the protein or the
degree of heterogeneity and decreases with the percent of glycosylation. As a general
guideline, one would start with approx 50–100 µg to profile the N-linked oligosaccharides
of a 60 kDa glycoprotein that contains 10–20% carbohydrate by weight. For O-linked
oligosaccharide analysis we suggest 100–500 µg of starting glycoprotein. This amount
would normally provide sufficient material for several electrophoretic runs. For isolation
of individual oligosaccharides, and carrying out sequencing, additional material will be
required.
3. The control for N-linked profiling consists of trypsin inhibitor that is used as a control for
enzyme digestion and fluorophore labeling. This control should be included in the analysis
for the following reasons:
a. If this control is used for the first time it will help the user to become familiar with the
procedures;
b. If the profile obtained looks appropriate then this assures the user that things are
working properly; and
c. In an unknown sample that may not contain N-linked oligosaccharides, the user can
be certain that the reagents are good and that the release and labeling procedures were
performed properly.
Similarly a control for O-linked profiling such as Bovine submaxillary mucin should be used.
4. Some proteins will precipitate when boiled, i.e., immunoglobulins. The following
procedure should be used if your protein precipitates: Add SDS/β-ME at the recommended
concentration and incubate for 5 min at room temperature, add NP-40 according to
directions, and then add PNGase F and incubate overnight.
5. The quantitation control consists of maltotetraose (Glucose 4). This control should be
prepared so that once prepared 5 µL will contain 200 pmol of maltotetraose (standards
prelabeled with ANTS are also available commercially). Accurate quantitation will be