Abstract
N-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 N-glycans has been extensively studied with all the major biosynthetic enzymes characterized. However, few studies have applied advanced mass spectrometry to profile intact plant N-glycopeptides. In this study, we use hydrophilic enrichment, high-resolution tandem mass spectrometry with complementary and triggered fragmentation to profile Arabidopsis N-glycopeptides from microsomal membranes of aerial tissues. A total of 492 N-glycosites were identified from 324 Arabidopsis proteins with extensive N-glycan structural heterogeneity revealed through 1110 N-glycopeptides. To demonstrate the precision of the approach, we also profiled N-glycopeptides from the mutant (xylt) of β-1,2-xylosyltransferase, an enzyme in the N-glycan biosynthetic pathway. This analysis represents the most comprehensive and unbiased collection of Arabidopsis N-glycopeptides revealing an unsurpassed level of detail on the micro-heterogeneity present in N-glycoproteins of Arabidopsis. Data are available via ProteomeXchange with identifier PXD006270.
Highlights
The N-glycosylation of proteins is a prevalent post-translational modification found in eukaryotes including microbes, animals and plants
Running Title: N-glycoproteome of Arabidopsis In this study, we report the analysis of tryptic N-glycopeptides derived from a microsomal membrane preparation of aerial tissues utilising hydrophilic interaction chromatography (HILIC) enrichment followed by high resolution tandem mass spectrometry (MS) employing complementary fragmentation techniques (HCD and electron-transfer dissociation (ETD)) to produce a robust and unbiased profile of N-glycopeptides from Arabidopsis
Since N-linked glycosylation occurs in the endomembrane, we isolated Arabidopsis microsomes (3,000 to 100,000 g) from 1 g FW of aerial tissues and digested proteins overnight with trypsin prior to enrichment of N-glycopeptides using HILIC SPE (Figure S2)
Summary
The N-glycosylation of proteins is a prevalent post-translational modification found in eukaryotes including microbes, animals and plants. The initial steps occur at the cytosolic side of the endoplasmic reticulum (ER) with dolichol phosphate (DolP) acting as the acceptor for the initial glycosylation steps to form a Man5GlcNAc2-DolP structure. Prior to entering the Golgi apparatus, the three Glc molecules and a single Man are removed in processes involving the calnexin (CNX) and calreticulin (CRT) cycle and ER quality-control (ERQC) processes resulting in a correctly folded glycoprotein with Man8GlcNAc2 glycan structures (Figure S1). Once in the Golgi apparatus, the Man residues are trimmed by mannosidases to form a Man5GlcNAc2 structure.
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