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Session_3b_Analytical_techniques - N-glycan analytical...

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N-glycan analytical methodology: an overview. Introduction: glycans as analytes. Carbohydrate structures with a typical composition (N-acetylglucosamine, mannose, galactose, glucose, fucose, sialic acid derivatives) pose specific problems towards analysis: 1) A very important issue is that these analytes do not strongly absorb light at wavelengths preferred for spectrophotometry (i.e. wavelenghts above 200 nm). Consequently, labeling of glycans is necessary to obtain sufficiently sensitive detection after their separation using a variety of techniques, as discussed below. The nature of the label can be chromophoric, fluorophoric or radioactive, depending on the detection system used. An alternative approach to the detection of N-glycans makes use of electrochemistry in pulsed amperometric mode (see below , see slides). 2) A second important feature of glycans is their strong hydrophilicity, due to a large number of hydroxylgroups in their structure. This is especially important with regard to their separation using chromatographic techniques, as most N-glycans are too hydrophilic to be sufficient retained on the more robust reversed phase resins. Improving their retention behaviour is another important reason for labeling with aromatic compounds. 3) Thirdly, an important subclass of the glycans is not charged at physiological pH, which poses a problem in the development of generally applicable electrophoretic methods, as electrophoresis requires the analytes to be charged in order to move in the electric field. This can be resolved by the use of strongly anionic tags, specially designed for electrophoresis. 4) A fourth point of importance is that glycan structure is inherently complex, mainly due to their branched nature, the less than 100% efficiency of most of the enzymatic reactions involved in their biosynthesis and the possibility in the biosynthetic pathway to generate isomeric structures with the same molecular weight and very similar behaviour. Thus, high separation resolution is a method feature which is highly appreciated in the field of glycan analysis. A problem which is not related with the physical properties of glycans, but which is of utmost importance in real-life glycobiological research, is that the analytes are very often only available
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in very minute quantities. In this respect, high sensitivity of any glycan analytical method makes the method more attractive. The term 'glycan analysis' can cover at least three distinct research goals: 1) Profiling: the high-resolution and high-sensitivity separation and detection of a mixture of glycans, where the obtained 'glycan profile' or 'fingerprint' contains sufficient information in itself to answer (part of) the research question. 2) The separation and subsequent detection and fractionation of a mixture of glycans, with the purpose of further structural characterization using other methods.
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