Solid-phase extraction of N-linked glycopeptides

Nat Protoc. 2007;2(2):334-9. doi: 10.1038/nprot.2007.42.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Deuterium
  • Glycopeptides / chemistry*
  • Glycopeptides / isolation & purification*
  • Glycosylation
  • Solid Phase Extraction / methods*
  • Succinic Anhydrides
  • Tandem Mass Spectrometry


  • Glycopeptides
  • Succinic Anhydrides
  • succinic anhydride
  • Deuterium