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