A New Strategy for Identification of N-Glycosylated Proteins and Unambiguous Assignment of Their Glycosylation Sites Using HILIC Enrichment and Partial Deglycosylation

TLDR

Mass spectrometry-based glycoprotein characterization spans from identifying carbohydrate–protein linkages to fully detailing all glycans at each glycosylation site. The study proposes a new strategy to pinpoint N‑glycosylation sites in complex samples by enriching glycopeptides with HILIC and partially deglycosylating them with endo‑beta‑N‑acetylglucosaminidases. After HILIC enrichment, the enzymes leave a single GlcNAc on asparagine, simplifying MS/MS spectra, and the method was validated on known glycoproteins and applied to lectin‑purified human plasma to map 62 sites in 37 proteins. The approach reduces spectral complexity and enables unambiguous site assignment, successfully identifying 62 N‑glycosylation sites across 37 glycoproteins in human plasma.

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.