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Nat Protoc. 2007;2(2):334-9.

Solid-phase extraction of N-linked glycopeptides.

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  • 1Institute for Systems Biology, Seattle, Washington 98103, USA.

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.

PMID:
17406594
PMCID:
PMC2971528
DOI:
10.1038/nprot.2007.42
[PubMed - indexed for MEDLINE]
Free PMC Article
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