title: Proteome-wide characterization of N-glycosylation events by diagonal chromatography.
abstract: A procedure to map N-glycosylation sites is presented here. It can be applied to purified proteins as well as to highly complex mixtures. The method exploits deglycosylation by PNGase F in a diagonal, reverse-phase chromatographic setup. When applied to 10 microL of mouse serum, affinity-depleted for its three most abundant components, 117 known or predicted sites were mapped in addition to 10 novel sites. Several sites were detected on soluble membrane or receptor components. Our method furthermore senses the nature of glycan structures and can detect differential glycosylation on a given site. These properties--high sensitivity and dependence on glycan imprinting--can be exploited for glycan-biomarker analysis.
authors.affiliation: Department of Biochemistry and Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B9000 Ghent, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Ghesquière
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Damme
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis*; D002845:Chromatography / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D040901:Proteomics / Q000379:methods*
chemical_list: D015415:Biomarkers; D006023:Glycoproteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase