OBJECTIVE: Quantitation and analyses of the fine structure of glycosaminoglycans are increasingly important for understanding many biological processes, including those most critical for understanding skeletal biology. We have developed a novel procedure, fluorophore-assisted carbohydrate electrophoresis (FACE), for determination of glycosaminoglycan fine structure and estimation of chain length. DESIGN: FACE utilizes enzymes that cleave glycosaminoglycans to create products, 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 for analysis of the glycosaminoglycans on aggrecan isolated from knee cartilage of 5- and 68-year-old patients, and assessment of hyaluronan oligosaccharides. CONCLUSIONS: The FACE technology is a powerful tool for analysis of all four classes of glycosaminoglycans obtained from a wide variety of biologic sources. 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.
OBJECTIVE: Quantitation and analyses of the fine structure of glycosaminoglycans are increasingly important for understanding many biological processes, including those most critical for understanding skeletal biology. We have developed a novel procedure, fluorophore-assisted carbohydrate electrophoresis (FACE), for determination of glycosaminoglycan fine structure and estimation of chain length. DESIGN: FACE utilizes enzymes that cleave glycosaminoglycans to create products, 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 for analysis of the glycosaminoglycans on aggrecan isolated from knee cartilage of 5- and 68-year-old patients, and assessment of hyaluronan oligosaccharides. CONCLUSIONS: The FACE technology is a powerful tool for analysis of all four classes of glycosaminoglycans obtained from a wide variety of biologic sources. 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.
Authors: Joanne C Krupa; David Shaya; Lianli Chi; Robert J Linhardt; Miroslaw Cygler; Stephen G Withers; John S Mort Journal: Anal Biochem Date: 2006-11-27 Impact factor: 3.365
Authors: Sabah N Rezvani; Jinnan Chen; Jun Li; Ron Midura; Valbona Cali; John D Sandy; Anna Plaas; Vincent M Wang Journal: J Orthop Res Date: 2019-09-13 Impact factor: 3.494
Authors: Frank S Cikach; Christopher D Koch; Timothy J Mead; Josephine Galatioto; Belinda B Willard; Kelly B Emerton; Matthew J Eagleton; Eugene H Blackstone; Francesco Ramirez; Eric E Roselli; Suneel S Apte Journal: JCI Insight Date: 2018-03-08