Journal
NATURE PROTOCOLS
Volume 2, Issue 2, Pages 334-339Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.42
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Funding
- DIVISION OF HEART AND VASCULAR DISEASES [N01HV028179] Funding Source: NIH RePORTER
- NATIONAL CANCER INSTITUTE [R21CA114852] Funding Source: NIH RePORTER
- NCI NIH HHS [R21 CA114852-03, R21 CA114852, R21-CA-114852] Funding Source: Medline
- NHLBI NIH HHS [N01-HV-8179, N01 HV028179] Funding Source: Medline
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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.
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