Protocol abstract
Nature Protocols 2, - 334 - 339 (2007)
Published online: 1 March 2007 | doi:10.1038/nprot.2007.42
Subject Categories: Isolation, purification and separation | Spectroscopy and structural analysis
Solid-phase extraction of N-linked glycopeptides
Yuan Tian1,2, Yong Zhou1, Sarah Elliott1, Ruedi Aebersold3 & Hui Zhang1,2
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.
- Institute for Systems Biology, Seattle, Washington 98103, USA.
- Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
- Swiss Federal Institute of Technology (ETH) Zurich and Faculty of Natural Sciences, University of Zurich, CH-8093 Zurich, Switzerland.
Correspondence to: Hui Zhang1,2 e-mail: hzhang32@jhmi.edu
nature-products
A-Z product listing
- (ESI)-qTOF) mass spectrometer(Waters)
- Affi-Prep beads(Bio-Rad Laboratories)
- BCA Protein Assay Kit(Pierce)
- C18 1cc SepPak columns(Waters)
- FAMOS autosampler(DIONEX)
- HPLC: HP1100(Agilent Technologies)
MORE ARTICLES LIKE THIS
These links to content published by NPG are automatically generated.
NEWS AND VIEWS
Mass spectrometry provides sweet inspirationNature Biotechnology News and Views (01 Jun 2003)