A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.
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.
Hägglund
Per
P
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Bunkenborg
Jakob
J
Elortza
Felix
F
Jensen
Ole Nørregaard
ON
Roepstorff
Peter
P
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D002853:Chromatography, Liquid; D006031:Glycosylation; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010949:Plasma / Q000737:chemistry*
D001216:Asparagine; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000117:Acetylglucosamine