A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation
Research output: Contribution to journal › Journal article › Research › peer-review
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-β-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 language | English |
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Journal | Journal of Proteome Research |
Volume | 3 |
Issue number | 3 |
Pages (from-to) | 556-566 |
Number of pages | 11 |
ISSN | 1535-3893 |
DOIs | |
Publication status | Published - 1 May 2004 |
- Endoglycosidase, Glycosylation, HILIC, Lectin affinity chromatography, Mass spectrometry, Plasma proteins, Post-translational modifications, Proteomics
Research areas
ID: 240161809