Most secreted proteins in eukaryotes are modified on the amino acid consensus sequence NxS/T by an N-glycan through the process of N-glycosylation. The N-glycans on glycoproteins are processed in the endoplasmic reticulum (ER) to different mannose-type N-glycans or, when the protein passes through the Golgi apparatus, to different complex glycan forms. Here we describe the capturing of N-glycopeptides from a trypsin digest of total protein extracts of Arabidopsis plants and release of these captured peptides following Peptide N-glycosidase (PNGase) treatment for analysis of N-glycan site-occupancy. The mixture of peptides released as a consequence of the PNGase treatment was analyzed by two dimensional nano-LC–MS. As the PNGase treatment of glycopeptides results in the deamidation of the asparagine (N) in the NxS/T site of the released peptide, this asparagine (N) to aspartic acid (D) conversion is used as a glycosylation ‘signature’. The efficiency of PNGase F and PNGase A in peptide release is discussed. The identification of proteins with a single glycopeptide was limited by the used search algorithm but could be improved using a reference database including deamidated peptide sequences. Additional stringency settings were used for filtering results to minimize false discovery. This resulted in identification of 330 glycopeptides on 173 glycoproteins from Arabidopsis, of which 28 putative glycoproteins, that were previously not annotated as secreted protein in The Arabidopsis Information Resource database (TAIR). Furthermore, the identified glycosylation site occupancy helped to determine the correct topology for membrane proteins. A quantitative comparison of peptide signal was made between wild type and complex-glycan-less (cgl) mutant Arabidopsis from three replicate leaf samples using a label-free MS peak comparison. As an example, the identified membrane protein SKU5 (AT4G12420) showed differential glycopeptide intensity ratios between WT and cgl indicating heterogeneous glycan modification on single protein.
- lectin affinity-chromatography
- glycosylated proteins
- linked glycoproteins