Comprehensive site- and structure-specific characterization of N-glycosylation in model plant Arabidopsis using mass-spectrometry-based N-glycoproteomics

The N-glycosylation is an important bioprocess in plant. Monosaccharide composition-level characterization at the intact N-glycopeptides has been extensively reported, yet structure-specific study to resolve multiple sequence structures of a single composition is still lacking. Here, we present a comprehensive structure-specific identification of intact N-glycopeptides of Arabidopsis with both HILIC and RAX enrichment, as well as GPSeeker and pGlyco database search. With target-decoy searches and spectrum-level FDR ≤ 1%, 5,687 N-glycopeptides from 3,713 N-glycosites of 3,140 N-glycoproteins were identified, which represents the currently most comprohensive profilling to our best knowledge. Wtih the experimental evidence support of structure-diagnostic fragment ions, 81 glycan structures from 54 glcan compostions were unambiguouly distinguished. The comprehensive experimental site- and structure-specific N-glycosylation data reported in this study will serve as a fundamental valuable reference for the coming functional studies of this widely adopted model organism of plant.