Mapping in vivo O-glycoproteome using site-specific extraction of O-linked glycopeptides (EXoO)

Protein glycosylation is one of the most abundant post-translational modifications. However, detailed analysis of in vivo O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, we present a chemoenzymatic method for the site-specific extraction of O-linked glycopeptides (EXoO), which enabled the unambiguous mapping of over 3,000 O-linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells and serum. This large-scale localization of O-linked glycosylation sites nearly doubles the number of previously identified sites, demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the ER and the Golgi. EXoO was also able to reveal significant differences in the in vivo O-linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.