*13.3. Analysis of Aggrecan's GAG Side Chains*

The CS chains of aggrecan are primarily of interest since these make a major contribution to aggrecans physicochemical and biological properties in tissues, whereas the function of the more minor KS chains are currently not known and thus are of lesser interest [116,233–235]. Several qualitative and quantitative methods have been developed for the discriminative measurement of intact GAG chains, including dye specific, thin layer chromatography (TLC), capillary electrophoresis [236–238], high-performance liquid chromatography (HPLC), various mass-spectrophotometric formats including liquid chromatography–tandem mass spectrometry (LC-MS/MS) [239–241], gas chromatography, enzyme linked immunosorption analysis (ELISA) using a wide array of anti-GAG antibodies, GAG microarrays and automated high-throughput mass spectrometric methods. Electrophoretic methods to separate intact GAG chains and GAG disaccharides generated by GAG depolymerising enzymes by capillary electrophoresis and conventional slab gel formats use media such as highly purified agaroses [242], polyacrylamide or mixtures of these as separation media. GAGs can be descriminated enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). These enzymes can be used in combination with chromatographic or electrophoretic separation methodologies to identify GAG species [243–247]. Following electrophoresis, electroblotting of the separated GAGs can be employed to nylon or nitrocellulose support membranes treated with cationic detergents. A large array of specific anti-GAG antibodies can be used for identification of the blotted GAGs. In gel, detection of separated

GAG disaccharides and oligosaccharide species can also be carried out using fluorophore assisted carbohydrate electrophoresis (FACE) [248]. Capillary electrophoresis, has high resolving power and sensitivity in the analysis of GAG composition, disaccharide sulphation patterns and sequence analysis [236–238].
