A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes.

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded.

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins.

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases.

Enzymatic deglycosylation followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples. Specific glycosidases were used to remove sugars from glycoproteins in a controlled fashion leaving the protein core intact; the resulting change in molecular weight could be detected as shifts in gel mobility. Alternatively, glycan-sensitive reagents were used to visualize the intensity of glycoprotein bands before and after enzyme treatment.

Multistage Tandem Mass Spectrometry of Chondroitin Sulfate and Dermatan Sulfate.

Chondroitin/dermatan sulfate (CS/DS) is a glycosaminoglycan (GAG) found in abundance in extracellular matrices. In connective tissue, CS/DS proteoglycans play structural roles in maintaining viscoelasticity through the large number of immobilized sulfate groups on CS/DS chains. CS/DS chains also bind protein families including growth factors and growth factor receptors.

Extraction of chondroitin/dermatan sulfate glycosaminoglycans from connective tissue for mass spectrometric analysis.

Chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) are present in high levels in connective tissue where they play roles as structural molecules and in protein-binding interactions. Recent developments in the techniques for analysis of CS/DS using capillary electrophoresis (CE) have enabled progress in the understanding of changes in CS/DS structure that accompany connective tissue diseases including osteoarthritis. Key to these developments is the ability to extract CS/DS GAGs from small quantities of connective tissue.

Historical overview of glycoanalysis.

More than half of all human proteins are glycosylated. Glycosylation defines the adhesive properties of glycoconjugates and it is largely through glycan-protein interactions that cell-cell and cell-pathogen contacts occur. Not surprisingly, considering the central role they play in molecular encounters, glycoprotein and carbohydrate-based drugs and therapeutics represent a greater than $20 billion market.