Chemical Glycoproteomic Profiling in Rice Seedlings Reveals N-glycosylation in the ERAD-L Machinery

As a ubiquitous and essential posttranslational modification occurring in both plants and animals, protein N-linked glycosylation regulates various important biological processes. Unlike the well-studied animal N-glycoproteomes, the landscape of rice N-glycoproteome remains largely unexplored. Here, by developing a chemical glycoproteomic strategy based on metabolic glycan labeling, we report a comprehensive profiling of the N-glycoproteome in rice seedlings. The rice seedlings are incubated with N-azidoacetylgalactosamine-a monosaccharide analog containing a bioorthogonal functional group-to metabolically label N-glycans, followed by conjugation with an affinity probe via click chemistry for the enrichment of the N-glycoproteins. Subsequent mass spectrometry analyses identify a total of 403 N-glycosylation sites and 673 N-glycosylated proteins, which are involved in various important biological processes. In particular, the core components of the endoplasmic reticulum-associated protein degradation machinery are N-glycosylated, and the N-glycosylation is important for the endoplasmic reticulum-associated protein degradation-L function. This work not only provides an invaluable resource for studying rice N-glycosylation but also demonstrates the applicability of metabolic glycan labeling in glycoproteomic profiling for crop species.