Abstract
Glycosaminoglycans (GAGs) are polysaccharides produced by most mammalian cells and involved in a variety of biological processes. However, due to the size and complexity of GAGs, detailed knowledge about the structure and expression of GAGs by cells, the glycosaminoglycome, is lacking. Here we report a straightforward and versatile approach for structural domain mapping of complex mixtures of GAGs, GAGDoMa. The approach is based on orthogonal enzymatic depolymerization of the GAGs to generate internal, terminating, and initiating domains, and nanoflow reversed-phase ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) tandem mass spectrometry (MS/MS) for structural characterization of the individual domains. GAGDoMa provides a detailed structural insight into the glycosaminoglycome, and offers an important tool for deciphering the complexity of GAGs in cellular physiology and pathology.
Highlights
Glycosylation is one of the most prevalent post-translational modifications of proteins and adds an immense and dynamic diversity to the proteome
The technology is based on orthogonal depolymerization of the chondroitin/dermatan sulfate (CS/DS) using primarily two bacterial lyases, chondroitinase AC and chondroitinase B, which cleaves between GalNAc and GlcA residues and GalNAc and IdoA residues, respectively, allowing distinction between IdoA-containing structures, DS motifs, and GlcA-containing structures, CS motifs, as well as internal, terminal, and linkage region domain structures
To challenge GAGDoMa, CS/DS primed on xylosides from two types of human cell lines were used as models as they were predicted to cover a wide range of structures[21,22]
Summary
Glycosylation is one of the most prevalent post-translational modifications of proteins and adds an immense and dynamic diversity to the proteome. Proteoglycans are one type of glycosylated proteins comprising one or more glycosaminoglycans (GAGs), which are extensively sulfated polysaccharides commonly composed of 25–100 repeating disaccharide units. To challenge GAGDoMa, CS/DS primed on xylosides from two types of human cell lines were used as models as they were predicted to cover a wide range of structures[21,22]. Using this technology, we have identified over 200 structures and characterized more than 60 of these in depth.
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