Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Carbohydrate-Containing Proteins
Methyl Glycoside Trimethylsilyl Method

In collaboration with J. Clamp (Z. Clamp)

The method was developed in the author's laboratory and is described in detail below.

Methanolysis. A 1.0 M solution of HCl in dry methanol, prepared by distillation over a Grignard reagent, is used. Dry gaseous HCl from a cylinder (BDH Ltd.) is bubbled through 250 mL of methanol placed in an ice bath. At a moderate gas flow rate, an HCl concentration of >1.0 mol/L is reached within 15 min; the solution is then diluted with dry methanol to a concentration of 1 mol/L. The molarity is usually determined by titrating an aliquot of the solution with sodium hydroxide. Next, the methanolic HCl solution is dispensed into 10-mL ampoules, which are purged with nitrogen, sealed, and stored at —20 °C.

The amount of glycoprotein required for the analysis is determined by its carbohydrate content and, ideally, amounts to 25–125 nmol of each monosaccharide. When studying a novel protein, 1–2 mg samples are typically used, which yields reliable results at an average carbohydrate content. A protein (glycoprotein) sample with added Internal Standards—mannitol (100 nmol) and perseitol (100 nmol)—is placed in a 2-mL ampoule and dried. To the dry residue, 0.5 mL of methanolic HCl is added, the mixture is purged with nitrogen, sealed, and heated for 24 h at 85 °C.

Upon completion of methanolysis, the mixture is neutralized with silver carbonate; its finely ground powder is added in small portions while monitoring the pH of the medium using narrow-range pH indicator paper.

N-Acetylation. Partial deacetylation of N-acetylhexosamines occurs during methanolysis. To block the liberated groups, additional acetylation is required, which is carried out by adding 0.1 mL of acetic anhydride to the neutralized reaction mixture; this simultaneously prevents the adsorption of sugars onto silver chloride-carbonate particles. The mixture is left to stand for 6 h and then centrifuged. The supernatant is transferred to a 5-mL pear-shaped flask. To the residue in the ampoule, 0.5 mL of methanol is added, mixed, centrifuged, and the supernatant is also transferred to the flask. The Procedure is repeated twice, and the combined solutions are evaporated at 35 °C. It is very important to thoroughly remove traces of acetic anhydride; therefore, if a characteristic odor persists, methanol is added to the residue once again and it is re-evaporated. After adding the third internal standard—arabitol (50 nmol in methanol)—the mixture is evaporated again with heating. Thus, the reaction flask contains the methyl esters of monosaccharide methyl Glycosides, internal standards—arabitol, mannitol, and perseitol—mixed with Some Amino Acids and Peptides resulting from the degradation of the polypeptide chain, which do not interfere with further analysis. At this stage, the dried sample can be stored in a vacuum desiccator over phosphorus pentoxide until analysis.

Trimethylsilylation. The free hydroxyl groups of sugars must be protected prior to gas Chromatography, and trimethylsilylation is the ideal procedure for this purpose. The reaction proceeds rapidly and quantitatively at room Temperature, and the Introduction of a large number of methyl groups leads to an increased detection sensitivity using a flame ionization detector. A mixture of trimethylchlorosilane — hexamethyldisilazane — pyridine (1:1:5) is used as the reagent. It is prepared weekly and stored in a tightly closed container. Upon standing, a small amount of ammonium chloride may form, which is removed by centrifugation before using the reagent. To the dry sample, 0.05 mL of the reagent mixture is added and left for 30 min at room temperature protected from moisture (a «dry» box can be used). Then the reaction mixture is centrifuged, and 1–5 µL of the supernatant is injected into the gas chromatograph.

Gas-Liquid Chromatography. Practical experience shows that most problems encountered when using the gas chromatography method are associated with failure to follow Column packing requirements. Here It is important to observe every instruction, no matter how minor. Glass columns measuring 2 m × 2–3 mm (internal diameter) are used. The packing is high-purity, acid-washed, silanized diatomite coated with 3–4% SE-30 phase. Suitable Materials are supplied by A number of companies. After injection, the temperature program is turned on from 120 to 210 °C at a rate of 1 deg/min.

To calculate the correction factors using internal standards, a mixture of Monosaccharides is passed through the column According to the full program. Neutral hexoses usually present no problems, but polar sugars—N-acetylhexosamines and sialic acids—act as true sensitive indicators of column performance. The sugar peaks must be strictly symmetrical; the appearance of small or broadened peaks indicates a poor column condition.

To condition the column for operation, the trimethylsilylation mixture is periodically passed through it for several hours under a slow nitrogen flow at 120 °C. Depending on the initial state of the column, this may take several days or even weeks; the process can be accelerated by passing standard sugar mixtures at defined intervals. Details of the method are described in works [25, 52, 67, 68, 70, 71].

5.22.3. Determination of uronic acids, hexosamines, and galactose of glycosaminoglycans by the methyl glycoside trimethylsilyl method [98]

Carbohydrate derivatives were prepared according to a modified procedure [70] (see also the previous section).

Gas-liquid chromatography. Fused silica column (25 m × 0.32 mm) with CP→Sil₅ liquid phase (Chrompak); flame ionization detector; carrier gas (N₂) flow rates: 0.7 mL/min, make-up gas to the detector: 30 mL/min; inlet temperature: 22 °C, detector temperature: 280 °C; temperature program from 135 to 215 °C, 1 deg/min. Separation of all pertrimethylsilyl derivatives of methyl glycosides, including arabitol, mannitol, mannose, glucose (2 peaks), and galactose (4 peaks), is achieved in less than 60 min. The yields of Monosaccharide Derivatives for all studied glycosaminoglycans are >87% (calculated per hexosamine).