An improved method for quantitative sugar analysis of glycoproteins

Abstract

Although there are numerous methods available to hydrolyze glycans utilizing strong acids, it all requires lengthy steps to obtain quantitative yield. We have developed a new simple one-step method for analysis of amino and neutral monosaccharides of glycoproteins quantitatively. Free monosaccharides were found to be stable during hydrolysis of glycans with 6 N HCl at 80 o C up to 2 h. Using this condition, analysis of free monosaccharides hydrolyzed from the bovine fetuin showed sugar composition of Gal : Man : GlcN : GalN = 13.2 : 11.0 : 15.5 : 2.6, which is closely matched with the reported value of 12.4 : 9.6 : 17.2 : 2.7 (Townsend et al., ABRF News 8: 14, 1997). This method was shown to be applicable to varieties of well-characterized glycoproteins, erythropoietin, fibrinogen and soybean agglutinin. The amounts of sugars released under the condition were very close to the experimental values by other procedures or to the theoretical ones. This condition was found to be suitable for direct sugar analysis of fetuin, which have been immobilized onto polyvinylidene difluoride membrane. Based on these results, it support that the 6 N HCl/80 o C/2 h is the simplest method for quantitative analysis of monosaccharide composition of glycoproteins.

Monosaccharide profiling of glycoproteins by capillary electrophoresis with contactless conductivity detection

“…However, it is important to note that the values of monosaccharide content found in different reports differ significantly. The values for Man, Glc, and Gal were slightly lower, (approximately two to three times) than average values reported in literature [29,33]. This can be considered as a reasonable agreement regarding the fact that the literature values also vary between different reports based on the type of hydrolysis, manufacturer of the fetuin sample, and variations can be found even between individual protein batches.…”

“…Fetuin was chosen as a model molecule, being a common glycoprotein that is often used in glycoproteomic studies as a standard because it has well-characterized glycan structures. Based on the literature [29], we chose HCl for acidic hydrolysis of fetuin. The standard of fetuin was hydrolyzed in a thermoblock at 50, 80 or 100 • C in 4 M HCl.…”

Metabolomics and13C Labelled Glucose Tracing to Identify Carbon Incorporation into Aberrant Cell Membrane Glycans in Cancer

“…LC-MS analysis revealed seven monosaccharides: PMP-glucose, PMP-galactose, PMP-mannose, PMP-L-fucose, PMP-glucosamine, PMP-galactosamine, and Neu5Ac (Figure 2B). Mammalian glycans do not contain hexosamines, 21 but because reaction with TFA removes the acetyl group in N-acetyl hexosamines leading to the formation of hexosamines, 22,23 we interpreted the presence of hexosamines as a surrogate for N-acetyl-hexosamines. Thus, these observations confirmed the unique identification of the primary monosaccharide components of membrane-bound glycans.…”

“…We asked whether the distributions of monosaccharides in the cell lines and the purified glycoprotein fetuin corresponded to predicted abundances based on previous studies. 22,23 We developed external standard curves of the derivatized monosaccharides to enable comparisons of the absolute levels of each monosaccharide (Supplementary Figure 3). The standard curves showed high precision and linearity in the measurements (R 2 > 0.996) and similar detection limits across the standards, confirming applicability to each of the monosaccharides measured in this study.…”

Monosaccharide profiling of silkworm (Bombyx mori L.) nervous system during development and aging

“…Monosaccharide determination was conducted as described in (Anumula 1994;Stepan and Staudacher 2011). Briefly, dry matter was redissolved in 30 lL of 4 N TFA (Trifluoroacetic acid, Sigma-Aldrich) per mg of dry matter and kept at 100°C for 4 h in order to release monosaccharides (Anumula 1994(Anumula , 2000Kim et al 2000;Harazono et al 2011). After acid hydrolysis, TFA was removed by vacuum concentrator (Martin Christ, RVC 2-25) at 30°C.…”