-linked Glycan Micro-heterogeneity in Glycoproteins of Arabidopsis.

-glycosylation is one of the most common protein post-translational modifications in eukaryotes and has a relatively conserved core structure between fungi, animals and plants. In plants, the biosynthesis of -glycans has been extensively studied with all the major biosynthetic enzymes characterized. However, few studies have applied advanced mass spectrometry to profile intact plant -glycopeptides. In this study, we use hydrophilic enrichment, high-resolution tandem mass spectrometry with complementary and triggered fragmentation to profile Arabidopsis -glycopeptides from microsomal membranes of aerial tissues. A total of 492 -glycosites were identified from 324 Arabidopsis proteins with extensive -glycan structural heterogeneity revealed through 1110 -glycopeptides. To demonstrate the precision of the approach, we also profiled -glycopeptides from the mutant () of β-1,2-xylosyltransferase, an enzyme in the -glycan biosynthetic pathway. This analysis represents the most comprehensive and unbiased collection of Arabidopsis -glycopeptides revealing an unsurpassed level of detail on the micro-heterogeneity present in -glycoproteins of Arabidopsis. Data are available via ProteomeXchange with identifier PXD006270.