Protein Protocols Protein Protocols

806 Hounsell, Davies, and Smith
9. 2 M Trifluoroacetic acid HPLC-grade.
10. 2 M HCl.
11. Dowex 50 W × 12 H + of cation-exchange resin.
12. 3.5-mL screw-cap septum vials (Pierce, Chester, UK) cleaned with chromic acid (2 L H 2SO 4/
350 mL H 2O/100 g Cr 2O 3) (Use care! extremely corrosive, see Note 1), and coated with
Repelcote (BDH, Poole, UK).
13. Teflon-backed silicone septa for 3.5-mL vials (Aldrich).
3. Method
1. Dry down the glycoprotein (10 µg) or oligosaccharide (1 µg) in a clean screw-top vial with
Teflon-backed silicone lid insert (see Note 2).
2. Hydrolyze in an inert N 2 atmosphere for 4 h at 100°C with 2 M HCl.
3. Dry the hydrolyzate and re-evaporate three times with HPLC-grade H 2O.
4. Purify on a 1-mL Dowex 50 W × 12 H + column eluted in water.
5. Dry down the monosaccharides ready for injection onto the HPLC system.
6. Prepare the following eluants:
Eluant A = 500 mL HPLC-grade H 2O.
Eluant B = 500 mL of 50 mM NaOH, 1.5 mM sodium acetate.
Eluant C = 100 mL of 100 mM NaOH.
7. Degas the eluants by bubbling through helium.
8. Place the postcolumn reagent in a pressurized reagent reservoir (300 mm NaOH) and
use the pneumatic controller to adjust helium pressure to give a flowrate of 1 mL/min
(approx 10 psi).
9. Equilibrate the column with 98% eluant A and 2% eluant B at a flowrate of 1 mL/min.
10. Add the postcolumn reagent between column and detector cell at a flowrate of 1 mL/min
via a mixing tee.
11. Inject approx 100 pmol of monosaccharide and elute isocratically as follows: eluant A = 98%;
eluant B = 2%; flowrate = 1 mL/min; for 30 min.
12. Calculate monosaccharide amounts by comparison with a range of known monosaccharide
standards run on the same day with deoxyglucose as an internal standard. From this,
it is possible to infer the type and amount of glycosylation of the glycoprotein.
13. Regenerate the column in eluant C for 10 min at 1 mL/min (see Note 3).
14. Re-equilibrate the column with 98%A/2%B before the next injection.
15. At the end of the analysis, regenerate the column in eluant C, and flush pumps with H 2O
(see Note 4).
4. Notes
1. If required, an equivalent detergent-based cleaner may be used.
2. Use polypropylene reagent vessels as far as possible for HPAEC-PAD because of the corrosive
nature of the NaOH, and to minimize leaching of contaminants from the reservoirs.
3. Some drift in retention times may be observed during the monosaccharide analysis. This
can be minimized by thorough regeneration of the column and use of a column jacket to
maintain a stable column temperature.
4. Failure to wash out the eluants from the pumps at the end of an analysis may result in
crystallization and serious damage to the pump heads.
References
1. Hounsell, E. F. (1993) A general strategy for glycoprotein oligosaccharide analysis, in
Methods in Molecular Biology, vol. 14: Glycoprotein Analysis in Biomedicine (Hounsell,
E. F., ed.), Humana, Totowa, NJ, pp. 1–15.