Fluorophore-assisted carbohydrate electrophoresis (FACE) of glycosaminoglycans

Objective: Quantitation and analyses of the fine structure of glycosaminogl ycans are increasingly important for understanding many biological processe s, including those most critical for understanding skeletal biology. We hav e developed a novel procedure, fluorophore-assisted carbohydrate electropho resis (FACE), for determination of glycosaminoglycan fine structure and est imation of chain length. Design: FACE utilizes enzymes that cleave glycosaminoglycans to create prod ucts, usually disaccharides, characteristic of the enzyme specificity. Each cleavage exposes a new reducing terminus that is fluorotagged by reductive amination with 2-aminoacridone. The tagged products are then displayed by electrophoresis, identified by their characteristic migration and chemistry , and quantitated by their molar fluorescence. Results: Each class of glycosaminoglycan and the enzymes specific for each class are discussed. Specific application of the FACE technology is shown f or analysis of the glycosaminoglycans on aggrecan isolated from knee cartil age of 5- and 68-year-old patients, and assessment of hyaluronan oligosacch arides. Conclusions: The FACE technology is a powerful tool for analysis of all fou r classes of glycosaminoglycans obtained from a wide variety of biologic so urces. While the FACE protocols are relative simple, they provide a wealth of information including quantitation in the pmole range, determination of fine structure, and estimation of chain length. (C) 2001 OsteoArthritis Res earch Society International.