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A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

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  • Per Hägglund
  • Jakob Bunkenborg
  • Felix Elortza
  • Ole Nørregaard Jensen
  • Peter Roepstorff
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Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.
Original languageEnglish
JournalJournal of Proteome Research
Volume3
Issue number3
Pages (from-to)556-66
Number of pages11
ISSN1535-3893
Publication statusPublished - 2004
Externally publishedYes

    Research areas

  • Acetylglucosamine, Amino Acid Sequence, Asparagine, Chromatography, Liquid, Glycosylation, Humans, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase, Mass Spectrometry, Molecular Sequence Data, Plasma

ID: 42362767