Site-specific Analysis of O-linked Glycoproteome Using Chemoenzymatic Strategy, part 2

Protein glycosylation is ubiquitous and plays critical roles in biology. However, study of O-linked glycoproteome (O-glycoproteome), a major type of protein glycosylation, has been severely impeded due to paucity of technology. We presented a chemoenzymatic strategy for extraction of site-specific O-linked glycopeptides (SOGO), which enabled simultaneous enrichment and identification of O-linked glycosylation sites (O-glycosites) in an unprecedented depth. Central to SOGO is a tandem-use of solid-phase capture of peptides and an O-linked glycan (O-glycan) dependent endoprotease named OpeRATOR. Interestingly, OpeRATOR requires O-glycan to specifically cleave the N-terminus of glycan-occupied Ser and Thr leading to unambiguous identification of O-glycosites and corresponding glycoproteins. The identified O-glycosites facilitated downstream determination of microheterogeneity of intact O-linked glycopeptides (O-glycopeptides). We benchmarked the method using human serum and identified 805 peptides containing 777 O-glycosites from 1,345 site-specific O-glycopeptides and 302 glycoproteins. We applied the method to study change of protein-specific O-glycosylation in human T cells infected with human immunodeficiency virus (HIV-1). Strikingly, 1404 peptides containing 1,352 O-glycosites from 577 glycoproteins were identified. In addition, altered site-specific O-linked glycosylation (O-glycosylation) during HIV infection of T cells were observed. In total, 1989 O-glycosites from 789 glycoproteins were precisely pinpointed from human serum and T cells. We observed conserved motifs for O-glycoproteome. Finally, ontology analysis revealed diverse roles for O-glycoproteins arguing broad application of our method to study biology in respective to protein O-glycosylation.