Fig. 1.

VAR2CSA affinity chromatography coupled to a glycoproteomics pipeline facilitates structural characterization of CS glycopeptides. CSPGs from human placenta, bladder tumor samples and two cancer cell lines were subjected to detergent extractions and fractionated on immobilized recombinant Var2CSA. The pull-down fractions were proteolytically digested with trypsin, and the generated CS glycopeptides were isolated through strong anion exchange (SAX) chromatography. The enriched CS-substituted glycopeptides were further subjected to chondroitinase ABC treatment to generate glycopeptides containing the CS linkage region. After reverse-phase chromatography on C18 column, the CS linkage region glycopeptides were analyzed by high-resolution mass spectrometry to fully characterize the core protein identities, the CS attachment sites and the glycan composition of the linkage region oligosaccharides.