Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Carbohydrate-Containing Proteins
Sensitive Methyl Glycoside Trimethylsilyl Method

A sample of glycoprotein (0.5–10 μg) or carbohydrate standard (0.1–1 nmol) is dried over P₂O₅ in vacuo in a 100 μL Reacti-Vial-type vessel. Meso-Inositol can be used as an internal standard. Dry HCl solution in methanol (40 μL, 0.625 mol/L) and methyl acetate (10 μL) are added. The vessel is sealed with a Teflon-lined cap, mixed on a vortex mixer, and incubated for 16 h at 70 °C. Cool. Add tert-butyl alcohol (10 μL) and dry in a stream of oxygen-free nitrogen.

N-Acetylation. To the residue, methanol (50 μL), pyridine (5 μL), and acetic anhydride (5 μL) are added sequentially with mixing, and the mixture is left for 15 min. Dry thoroughly first in a stream of nitrogen and then in vacuo over P₂O₅. Silylation is carried out by adding 20 μL of silylating agent (Sylon HTP, Supelco). Mix on a vortex mixer and leave for 1 h at room Temperature. Dry. The residue is immediately dissolved in 14 μL of hexane, and the entire sample is loaded onto the Gas-Liquid Chromatography Column.

Gas-liquid chromatography. A WCOT fused-silica capillary column (25 m × 0.32 mm) with a CP-Sil 5 liquid phase (Chrompak); temperature program: 140 °C for 12 min, followed by heating at a rate of 8 °C/min to 260 °C; flame ionization detector.

Separation of 29 TMS derivatives of neutral sugars, amino sugars, and acids was achieved in less than 16 min. Sensitivity is <100 pmol for each peak.