Alkaline β-elimination

Release of glycans by alkaline β-elimination utilizes ammonium hydroxide/carbonate or sodium hydroxide in conjunction with sodium borohydride. Like hydrazinolysis, β-elimination results in total destruction of the protein backbone.1-3

O-Glycosidic linkages between glycans and the β-hydroxyl groups of serine or threonine are readily hydrolyzed by dilute alkaline solutions (0.05 to 0.1 M sodium hydroxide or potassium hydroxide) under mild conditions (45 to 60 oC for 8 to 16 hours) leading to the liberation of Olinked glycans by the mechanism of β-elimination. To prevent isomerization or degradation of the released carbohydrates by ′peeling′ reactions, the hydrolysis is performed in the presence of a reducing agent (0.8 to 2 M sodium borohydride). This results in the formation of the reduced (alditol) forms of the glycans. N-Linked glycans and O-linked glycans that are attached to tyrosine, hydroxyproline and hydroxylysine are not cleaved under these conditions. Furthermore, the β-elimination reaction does not take place if the glycan is attached to serine orthreonine at the carboxy-terminus position of the protein.

For quantitative release of N-linked glycans, harsher alkaline conditions are required (1 M sodium hydroxide at 100 °C for 6 to 12 hours). As for O-linked glycans, the reaction must be performed under reducing conditions (1 to 2 M sodium borohydride) to prevent ′peeling′ reactions taking place on the released N-glycans. N-Acetylglucosamine (GlcNAc) residues are deacetylated during this reaction and must be re-Nacetylated by using acetic anhydride in methanol during the recovery ofthe glycans.

The primary limitation of alkaline β-elimination is the high residual sodium content after reaction. The high salt content interferes with downstream mass spectrometry techniques for glycan mapping and sequencing. An alternative method for β-elimination uses ammonia in place of hydroxide for the high pH conditions required to cleave the Olinked glycan residues. However, β-elimination using ammonia may be incomplete for some proteins.4

A 5 mg/ml solution of borate-ammonia complex in 28% aqueous ammonia is used to hydrolyze the O-glycosidic bonds. Glycoproteins are incubated at 45 oC for 18-24 hours with the borate-ammonia complex solution to release the O-linked glycans. Excess ammonia is removed by flushing the sample with nitrogen until dry. The sample is reconstituted with water and passed through a cation exchange resin such as Dowex® 50W×8. Excess boric acid is removed by repeated reconstitution with methanol and evaporation using nitrogen. The released glycans can be redissolved in water and labeled for mass spectrometric analysis without desalting.5-6
For more information on Glycobiology please browse our online Glycobiology Analysis Manual.
References:
1. Patel, T.P. and Parekh, R.B., Release of oligosaccharides from glyco­proteins by hydrazinolysis. Meth. Enzymol., 230, 57-66 (1994).
2. Patel, T., et al., Use of hydrazine to release in intact and unreduced form both N- and O-linked oligo­saccharides from glycoproteins. Biochemistry, 32, 679-693 (1993).
3. Bendiak, B., and Cumming, D.A., Purification of oligosaccharides having a free reducing-end from glycopeptide sources. Carbohydr. Res., 151, 89-103 (1986).
4. Makino, Y., et al., Structural analysis of N-linked sugar chains of human blood clotting factor IX. J. Biochem. (Tokyo), 128, 175-180 (2000).
5. Takasaki, S., et al., Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides. Meth. Enzymol., 83, 263-8 (1982).

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A6404 Acetic anhydride reagent grade, ≥98%
287717 Borane-ammonia complex technical grade, 90% Green Alternative
217514 Dowex® 50WX8 hydrogen form hydrogen form, 200-400 mesh
PP0540 GlycoProfile β-Elimination Kit
213462 Sodium borohydride ReagentPlus®, 99%
S5881 Sodium hydroxide reagent grade, ≥98%, pellets (anhydrous)