Protein Protocols Protein Protocols

HPLC of Fluorescently Labeled Glycans 875
Fig. 3. Analysis of fetuin on Weak Anion Exchange Chromatography. The separation of
glycans possessing from 1 to 5 sialic acids is shown.
3. Sample loading:
a. The sample should be loaded as an aqueous solution. A volume of 95 µL of sample
should be loaded.
b. No suitable standards are currently available although it is useful to run a well characterized
glycoprotein such as fetuin.
c. The glycans generally elute on the basis of charge although the size of the glycan also
contributes to the position of elution.
d. A typical separation of charged N-glycans from bovine serum fetuin is shown in Fig. 3.
3.6. Exoglycosidase Digestion of N-Glycans
Digestions are generally performed on the pool of glycans by the application of
exoglycosidases under the incubation conditions shown in Table 1 in a series of
enzyme arrays of increasing complexity as shown in Table 2.
1. The amount required for each digestion can be judged from the previous profiling run, but
in general 100 fmol of glycan is detectable. In practice more may be used with the enzyme
concentrations given below if sufficient material is available.
2. Pipet no more than 50 µL of solution into a 0.5-mL microcentrifuge tube.
3. Evaporate glycan solutions to dryness in a vacuum centrifuge.
4. Add 2–5 µL of enzyme solution and 2 µL of incubation buffer.
5. Make the total volume up to 10 µL with water.
6. Incubate for 16–24 h at 37°C.
7. Cool and load the digestion mixture onto a protein binding filter (Microspin 45).
8. Leave for 15 min at room temperature.
9. Centrifuge for 15 min at 5000g.
10. Wash tube with 10 µL of freshly prepared 5% acetonitrile in water and load onto filter.
11. Leave for 15 min at room temperature.
12. Centrifuge for 15 min at 5000g.
13. Repeat steps 10–12.
14. Twenty microliters of sample may be directly analyzed by HPLC if sufficient material is
available; if not, then dry down sample and redissolve in 20 µL of water.
15. An example of digestion of N-glycans from IgG is shown in Fig. 4.