Application of the 50% Hydrazine Solution Method for O-Glycans Release, their Chemical Labeling, and HPLC Separation

Figures & data

Table 1 Monosaccharide composition of pig gastric mucin glycopeptide preparation

Monosaccharide	Fucose	Mannose	Galactose	GalNAc	GlcNAc
Amount of monosaccharide (μmol)	0.146	0.008	0.271	0.100	0.279
Approximate mol ratio to GalNAc	1.5	0.1	2.7	1	2.8
Mass percentage (%)	15	1	31	14	39

A sample of isolated glycopeptide was submitted to methanolysis and reacetylation followed by silylation, and analyzed for its monosaccharide composition by gas chromatography.

Table 2 Comparison of neutral carbohydrate recovery after subsequent steps of oligosaccharide isolation

	TEA/N2H4	NaOH/NaBH4	NaOH/N2H4
1. Content of neutral carbohydrates in starting glycopeptide (μg)	625	625	625
2. Content of neutral carbohydrates in β-elimination products (μg)	510	350	260
Recovery of neutral carbohydrates after β-elimination (2:1)	81.5%	56%	41.5%
3. Content of neutral carbohydrates in undermembrane fraction (μg)	300	281	190
Recovery of oligosaccharides from β-elimination products (3:2)	59%	80%	73%
Recovery of oligosaccharides from starting glycopeptide (3:1)	48%	45%	30%

Standardized samples of glycopeptide were submitted to oligosaccharide releasing procedure according to the hydrazine method (TEA/N₂H₄), to the classical reductive β-elimination (NaOH/NaBH₄), and to a modified hydrazine procedure with NaOH instead of 3-ethylamine (NaOH/N₂H₄). Neutral carbohydrates were determined according to the method of Dubois et al. (1966).

FIGURE 1 Gas chromatographic separation of lactose constituents. The gray line represents three peaks of galactose and two peaks of glucose, all of untagged lactose origin, and internal standard, mannitol. The black line represents lactose after labeling with p-aminobenzoic acid ethyl ester (ABEE).

FIGURE 2 HPLC separation of ABEE-labeled mucin oligosaccharides on a DEAE column. N, neutral oligosaccharide fraction; I and II, acidic oligosaccharides. Neutral oligosaccharides were pooled, desalted on LC18 SPE cartridges, condensed, and submitted to rechromatography on amine phase column.

FIGURE 3 Separation of ABEE-labeled neutral oligosaccharides from pig gastric mucin on LiChrospher 100 NH₂ HPLC column. Numbered peaks were analyzed for their monosaccharide composition (Table 3).

Table 3 Monosaccharide analysis of peaks eluted from the LiChrospher NH₂ column

	Contents of each monosaccharide in each peak (μmol/peak)
Number of peaks	Fuc	Gal	Glc	GalNAc	GlcNAc
6	0.045	0.074	0.002	0.005	0.065
7	0.055	0.063	0.025	0.005	0.050
8	0.062	0.051	—	Trace	0.031
9	0.016	0.030	0.004	Trace	0.011
11	0.020	0.041	0.008	0.005	0.043

Released monosaccharides were fractionated on a DEAE column and the neutral oligosaccharide pool was fractionated on the LiChrospher NH₂ column. Some of the obtained peaks from the LiChrospher column were pooled and submitted to gas chromatographic analysis for their monosaccharide composition. Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine.