1 Department of Systems Biology, Technical University of Denmark2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark3 Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark4 Proteomics Platform, Department of Systems Biology, Technical University of Denmark5 Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark
The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications. Glycosylation is one of the most common PTMs of protein that is involved in many physiological functions and biological pathways. The aim of this Ph.D. project is mainly to screen and identify N-glycosylated proteins from barley and wheat. A HILIC-based glycopeptide enrichment technqiue was first developed by supplementing cotton wool with ZIC-HILIC in a microcolumn (called ZIC-cotton). This approach reduced co-enrichment of non-glycosylated peptides and allowed glycoppeptide identification from large protein mixtures. It was applied for glycoprotein identification and glycosylation site assignment in wheat albumin and barley aleurone layer proteins. By sitespecific glycosylation labeling and LC-MS/MS analysis, 76 different glycosylation sites within 65 wheat albumin proteins were identified using a combination of ZIC-cotton and cotton wool. In addition, ZIC-cotton has been also applied to proteins produced from barley aleurone layer and 47 glycoproteins were identified. Sequence homology search against allergen database reveals that many glycoproteins identified from wheat and barley share similarity with known food allergens and may therefore be targets in search of novel allergens from wheat flour.