Glycosaminoglycans (GAGs) of the chondroitin sulfate (CS) class are composed of anionic polysaccharide chains that are O-glycosidically linked, via a linkage region, to specific Ser residues of extracellular glycoproteins to form the CS proteoglycans (PGs). CSPGs are widely distributed in the extracellular matrix (ECM) and on cell surfaces. CSPGs are involved in many aspects of cell signalling and in organism development; and are major building blocks of cartilage and bone. A major human CSPG is aggrecan, composed of 2530 amino acid residues potentially carrying >100 CS chains, which is an important structural constituent of cartilage. Significantly, protease degradation of aggrecan leading to a loss of cartilage is a key feature in the development of osteoarthritis. To better understand the structure, function and biomedical importance of expressed and post-translationally modified ECM proteins, the matrisome, it is crucial to structurally characterize and quantitate them.

In the emerging field of glycoproteomics, N- and O-glycopeptides are LC-MS/MS analysed, after protease digestion and enrichment procedures, to facilitate the site-specific glycan analysis of glycoproteins. In line with this, we have developed glycoproteomic methods to investigate the linkage region glycan structure of CSPGs and to identify novel CS-glycosites from human urine samples. Since children have a relatively high concentration of urine GAGs we have now started to analyse urine samples from 0.5-19 year old children. Thereby a multitude of endogenously cleaved CS-glycopeptides from aggrecan were identified that were not present in urine samples from adults. Two examples are S.GLPS.G and G.VEDIS.G. By using the Byonic software, 719 unique glycopeptides from 231 different peptides encompassing up to 151 out of the 184 possible SG and SA CS-glycosites of aggrecan were identified. Thus, thanks to the presence of the endogenously cleaved CS-glycopeptide fragments in the urine from children we were able to characterize the site-specific CS glycosylation of aggrecan that previously had been inaccessible for analysis.

Several new LC-MS/MS approaches were used for the structural analyses. For instance, fragmentation analysis after negative mode higher-energy collision dissociation (HCD) was used to characterize the sulfate distribution pattern of the GAG chains of the dominating GLPS glycopeptides. The presented methodologies will be of importance in future CS glycoproteomics projects, and it will be important to investigate whether the aggrecan glycopeptides can be used as biomarkers in adults, for instance in osteoaerthritis or in reumatic disorders.