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Ammonium-based alkali-catalyzed [beta]-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with [beta]1,3-1inked cores. In contrast to the previously studied [beta]1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the [beta]l,3-linkaga is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages ([alpha]l,2-, [beta]1,2-, [alpha]l,3-,[beta]1,3-, [alpha]l,4-, [beta]1,4-, [alpha]1,6-, and [beta]l,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O-and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O-and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glyean release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies. 10.1021/ac102300r

The glycosaminoglycan (GAG) family of biomacromolecules is composed acidic and linear chains of repeating disaccharide units. Quantitative disaccharide composition analysis is essential for the study and characterization of GAGs. Heparan sulfate and heparin consist of multiple disaccharide units and can be well-separated by ion-pairing reversed-phase microflow high-performance liquid chromatography (IPRP-Mf-HPLC). Each disaccharide can be detected and its mass confirmed by electrospray ionization mass spectrometry (ESI-MS). Isotopically enriched disaccharides were prepared chemoenzymatically from a uniformly [sup.13]C,[sup.15]N-labeled N-acetylheparosan (-GlcA(I[right arrow]4)GlcNAc-) obtained from the fermentation of E. coli K5. These isotopically enriched disaccharides have identical HPLC retention times and mass spectra as their unlabeled counterparts and were used in liquid chromatography--mass spectrometry (LC--MS) as internal standards. The ratio of intensifies between each pair of enriched and nonenriched disaccharides showed a linear relationship as a function of concentration. With the use of these calibration curves, the amount of each disaccharide ([greater than or equal to] 2 ng/ disaccharide) could be quantified in four heparan sulfate samples analyzed by this method.

Monosaccharide analysis is a critical way to profile the composition of complex carbohydrates. Methods to analyze neutral and amino sugars have been established for a long time, but methods for acidic sugars are rare. The acidic sugars, including uronic acids and sialic acids, are also important components in some complex carbohydrates. In this report, a high-performance anion-exchange chromatography method with pulsed amperometric detection was initially developed to analyze acidic sugars including different uronic acids and sialic acids. Subsequently, a method to profile complete monosaccharides, including most neutral, amino, and acidic sugars, was developed. This method has a limit of quantitation of ~12.5 × 10-3 nmol for each sugar as well as good linearity over a wide range. This is a convenient procedure because it avoids additional derivatization of monosaccharides and has a broad application to a wide range of complex carbohydrates. The monosaccharide compositions of a variety of complex carbohydrates such as different glycosaminoglycans, alginate, fucoidan, and glycans were profiled by this comprehensive method. In addition, the hydrolysis patterns of these complex carbohydrates are discussed. [ABSTRACT FROM AUTHOR]

Low molecular weight heparins (LMWHs) are polydisperse and microheterogenous mixtures of polysaccharides used as anticoagulant drugs. Profiling analysis is important for obtaining deeper insights into the structure of LMWHs. Previous oligosaccharide mapping methods are relatively low resolution and are unable to show an entire picture of the structural complexity of LMWHs. In the current study a profiling method was developed relying on multiple heart-cutting, two-dimensional, ultrahigh performance liquid chromatography with quadruple time-of-flight mass spectrometry. This represents an efficient, automated, and robust approach for profiling LMWHs. Using size-exclusion chromatography and ion-pairing reversed-phase chromatography in a two-dimensional separation, LMW components of different sizes and LMW components of the same size but with different charges and polarities can be resolved, providing a more complete picture of a LMWH. Structural information on each component was then obtained with quadrupole time-of-flight mass spectrometry. More than 80 and 120 oligosaccharides were observed and unambiguously assigned from the LMWHs, nadroparin and enoxaparin, respectively. This method might be useful for quality control of LMWHs and as a powerful tool for heparin-related glycomics.