Glycosylation is a common post translational modification of proteins which adds a complex carbohydrate moiety to one or more sites of the protein. These modifications are found in a large proportion of human proteins and play important roles in organ development, cancer growth, and viral infection mechanisms. Despite their biomedical relevance, methods understanding molecular basis for glycoprotein functions is often hampered by the heterogeneity of the attached glycans. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are complementary methods that have much to offer for the structural analysis of these challenging targets. NMR spectroscopy allows for atomic level resolution and can assess the internal dynamics of macromolecules.
This presentation will discuss advancements in NMR resonance assignment of glycoproteins produced with sparse isotope labels. These methods are then applied to better understand the domain orientation of the glycosaminoglycan-binding glycoprotein Roundabout 1. The use of a construct engineered with a lanthanide binding tag allows for structural analysis. In a separate application, top-down mass spectrometry is used to measure glycan occupancy of a model protein and gain insight into the mechanism of OST-B catalyzed N-glycosylation.