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 Denmark6 University of Southern Denmark
Hydrophilic liquid chromatography (HILIC) is used extensively as a sample preparation step for glycopeptide enrichment in proteome research. Here, we have applied cotton wool and a zwitterionic HILIC (ZIC-HILIC) resin in solid-phase extraction microcolumns to provide a higher loading capacity and broader specificity for glycopeptide enrichment. This strategy was applied to tryptic digests of wheat flour albumin extracts followed by simulataneous site-specific 18O labeling and deglycosylation using peptide-N-glycosidase A (PNGase A) in H218O. Subsequent LC–MS/MS analysis allowed for assignment of 78 N-glycosylation sites in 67 albumin proteins. Bioinformatic analysis revealed that several of the identified glycoproteins show sequence similarity to known food allergens. In addition, the potential impact of some of the identified glycoproteins on wheat beer quality is discussed.
Journal of Proteome Research, 2014, Vol 13, Issue 5, p. 2696-2703