NMR Analysis of Glycans

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Nuclear magnetic resonance (NMR) spectroscopy measures the extent to which a glycan or other molecule distorts a magnetic field. This technique is non-destructive and can provide full structural information about the glycan being analyzed. The traditional approach to structural characterization of a pool of free glycans by NMR spectroscopy has been to purify each component for individual analysis. By using a combination of one-dimensional proton and carbon NMR spectra and two-dimensional homonuclear and heteronuclear NMR methods, the ratios of monosaccharides present and ratios of their anomeric bonds can be assigned.¹ Nano-NMR analysis using high resolution spectrometers has been shown to be suitable for structural analysis of limited amounts of glycan samples as well as mixtures of N-linked and O-linked glycans.² NMR methods that take advantage of fast exchange are suitable for studying glycan-protein interactions.^3,4

Calculating the chemical shifts of known glycans with precision is laborious. Databases that are compiled from experimentally determined NMR data of known glycans allow rapid comparison of spectra and structure assignments for unknown glycans. Publicly available databases created by glycomics organizations contain characteristic NMR resonance (chemical shifts) and coupling constants for a number of glycans. The German Cancer Research Centre in Heidelberg, Germany, has developed and hosts the GlyNest NMR shift estimation program as part of their Internet-based portal (www.GLYCOSCIENCES.de).⁵ GlyNest uses a spherical environment encoding scheme to estimate the chemical shifts of glycans using an online database. Alternatively, CASPER is a program developed and hosted by Stockholm University in Stockholm, Sweden; it can be accessed either directly or through the GLYCOSCIENCES.de website. CASPER uses an incremental rule-based approach to calculate chemical shifts of the free reducing monosaccharides adjusted for the attached carbohydrates in proximity. When compared by Loβ, et al., the two services were found to calculate ¹H- and ¹³C-NMR chemical shifts of glycans with sufficient precision to be suitable for assignments of structure based on NMR spectra.⁶

Electrophoresis of Glycans

Separation of glycans by electrophoresis in polyacrylamide gel has been widely used and different methods are described in the literature for analysis of monosaccharides and oligosaccharides. The most commonly used system is the electrophoresis of fluorophore-labeled glycans in highly cross-linked polyacrylamide gels and is termed as Fluorophore- Assisted Carbohydrate Electrophoresis (FACE). The glycans are usually labeled with a fluorscent tag, mainly ANTS or AMAC, and separated on 20‑40% gels. The extent of cross-linking means that extra precautions should be taken to prevent heating and warping of the gel during the run. After electrophoresis, the band patterns are visualized by illuminating the gel under UV light and photographing the image. Although this technique is sensitive in the sub-picomolar range, the resolution between the glycans can be poor due to the limitation on the size of the gel. Figure 1 shows the mobility of four individual N-linked glycans after labeling with ANTS. This method can also be used to obtain profiles of glycans released from glycoproteins and to elucidate the structure of an individual glycan after exoglycosidase digestion, based on the mobility shift of the parent glycan band.