Functional Glycomics Methods and Protocols
The study of functional glycomics requires the continuous development of rapid and sensitive methods for the identification of glycan structures and integration to structure-function relationships. In Functional Glycomics: Methods and Protocols, a panel o
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Introduction Determination of the functional role of carbohydrates in biology requires the development of methodologies that can analyze carbohydrate structures with increasing levels of sensitivity and simplicity. Recent efforts by our laboratory to characterize the glycome of a number of important bacterial pathogens led to the elucidation of many surface glycan structures (LOS, CPS, N-linked, and O-linked glycoproteins) and the development and J. Li (ed.), Functional Glycomics, Methods in Molecular Biology 600, DOI 10.1007/978-1-60761-454-8 11, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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adaptation of rapid methods for glycan detection and study of carbohydrate–protein interactions (1–7). The presence of novel monosaccharides in bacterial glycans presents unique challenges for structural analysis. Mass spectrometry plays an integral part in the discovery of these novel glycans due to its high sensitivity and mass accuracy. With recent advances in NMR technology, detailed structural characterization from nanomolar amounts of purified glycans is now possible (Fig. 11.1). Of all the spectroscopic methods currently available, high-resolution NMR spectroscopy offers the most complete approach for structure determination of saccharides which includes the determination of the stereochemistry of sugars, sequence, non-carbohydrate substituents, as well as conformation and dynamics. In addition, high-resolution NMR can facilitate the study of sugar–protein interactions. Another advantage of NMR is that labile compounds can be analyzed without purification of individual molecules, a process which can often lead to the loss of labile components. In comparison with mass spectrometry, NMR is, however, a relatively insensitive technique in terms of the amount of material required. Recent advances in equipment and continuing developments in techniques are reducing the threshold in the amount of material required. Functional glycomics of bacterial glycoproteins requires the identification of novel glycans on glycoproteins and the genes associated with their synthesis. One approach is to complete the structural characterization of a novel glycan and to use this information to analyze bioinformatically the corresponding genome
Fig. 11.1. Increase in signal to noise obtained using a cryogenically cooled probe. The anomeric region of the NMR spectrum for a sugar metabolite sample (200 L 99% D2 O) isolated from C. jejuni 81–176 (8) is shown. (a) 1 H NMR spectrum (128 scans) acquired at 600 MHz using a cold probe (S/N = 100:1). (b) 1 H NMR spectrum (128 scans) acquired at 500 MHz using a standard probe (S/N = 39:1).
NMR Spectroscopy to Functional Glycomics
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to identify putative glycan biosynthetic genes which share homology with genes responsible for the biosynthesis of similar sugars in other organisms. These genes can then be mutated to confirm their role and/or the encoded proteins expressed recombinantly for detailed functional characterization and elucidation of biosynth
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