N-glycoproteome profiling of rice

Metabolic glycan labeling (MGL) is a technique that utilizes bioorthogonal chemical reporters to investigate protein glycosylation. While this method has been widely used for glycoproteomic profiling in animals, its application in N-glycoproteomic research in crops remains limited and requires further development. In this study, we employed N-azidoacetylgalactosamine (GalNAz), a click-compatible sugar, to conduct a large-scale analysis of the N-glycoproteome in rice. Using this approach, we successfully identified 426 N-glycosylation sites and 680 N-glycosylated proteins involved in various important biological processes such as metabolism and stress response. A subset of randomly selected proteins identified by mass spectrometry were validated and confirmed to be N-glycosylated, supporting the reliability of the MGL-based method for N-glycosylation identification in rice. Moreover, by combining mass spectrometry data with biochemical validation, we discovered that the core components of the ERAD-L machinery undergo N-glycosylation. Importantly, this N-glycan modification of the ERAD-L machinery is conserved across both plants and animals. Functional studies revealed the crucial role of N-glycosylation in proper protein degradation within the ERAD-L machinery. Overall, our research significantly expanded the database of N-glycoproteins in rice and presented a highly efficient and practical approach for large-scale identification of N-glycosylated proteins, which holds potential applications for identifying N-glycosylated proteins in other crops.