N- and O-Glycoproteomic Analysis of Complex Proteoglycans

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are also N- and O-glycosylated. All of these types of glycosylation are crucial to their function, but present a considerable analytical challenge. Using serial proteolysis and electron capture/higher energy collisional dissociation (EThcD) we seek to improve the sequence coverage of several complex proteoglycans. In many cases, the use of HCD alone allows for the identification of more glycopeptides, but EThcD allows for the confident assignment of glycan compositions on multiply glycosylated peptides. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples.