Solid-phase extraction of N-linked glycopeptides

Yuan Tian, Yong Zhou, Sarah Elliott, Ruedi Aebersold, Hui Zhang

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)334-339
Number of pages6
JournalNature protocols
Volume2
Issue number2
DOIs
StatePublished - Mar 2007

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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