title: Solid-phase extraction of N-linked glycopeptides.
abstract: Protein glycosylation is a common post-translational modification and has been increasingly recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. N-linked glycosylation is prevalent in proteins on the extracellular membrane, and many clinical biomarkers and therapeutic targets are glycoproteins. Here, we describe a protocol for solid-phase extraction of N-linked glycopeptides and subsequent identification of N-linked glycosylation sites (N-glycosites) by tandem mass spectrometry. The method oxidizes the carbohydrates in glycopeptides into aldehydes, which can be immobilized on a solid support. The N-linked glycopeptides are then optionally labeled with a stable isotope using deuterium-labeled succinic anhydride and the peptide moieties are released by peptide-N-glycosidase. In a single analysis, the method identifies hundreds of N-linked glycoproteins, the site(s) of N-linked glycosylation and the relative quantity of the identified glycopeptides.
authors.affiliation: Institute for Systems Biology, Seattle, Washington 98103, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.forename: Sarah
authors.initials: S
authors.lastname: Elliott
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites; D003903:Deuterium; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D006031:Glycosylation; D052616:Solid Phase Extraction / Q000379:methods*; D013387:Succinic Anhydrides; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D013387:Succinic Anhydrides; C031801:succinic anhydride; D003903:Deuterium