N-glycosylation of plant proteins has been shown to be important for protein folding, correct subcellular location and secretion, enzyme catalytic activity as well as plant pathogen interactions¹. At the modification site there is (micro) heterogeneity of the glycans attached, making it difficult to detect in routine proteomics studies and the labile nature of the glycan hinders the analysis by mass spectrometry (MS). Plant N-glycosylation profiling studies performed have primarily relied on removal of the N-glycans to ease both the identification of the peptide and the glycan but at the cost of losing information on site-specific microheterogeneity².

Current MS-based ionisation and fragmentation techniques allow the comprehensive sequencing of intact glycopeptides revealing both the peptide and glycan sequences as well as the N-glycosylation site position. We describe a method for the enrichment of N-linked glycopeptides from the model plant Arabidopsis, followed by LC-ESI-MS/MS analysis using higher-energy collision dissociation product dependent electron transfer dissociation (HCD-pd-ETD) fragmentation for peptide identification, glycan site attachment and glycan sequence.