A Chemoenzymatic Method for Glycoproteomic N-glycan Type Quantitation

Abstract

29Glycosylation is one of the most important post-translational modifications in biological systems. 30Current glycoproteome methods mainly focus on qualitative identification of glycosylation sites or intact 31 glycopeptides. However, the systematic quantitation of glycoproteins has remained largely unexplored. 32Here, we developed a chemoenzymatic method to quantitatively investigate N-glycoproteome based on 33 the N-glycan types. Taking advantage of the specificity of different endoglycosidases and isotope 34 dimethyl labeling, six N-glycan types of structures linked on each glycopeptide, including high-35 mannose/hybrid, bi-antennary and tri-antennary with/without core fucose, were quantified. As a proof of 36 principle, the glycoproteomic N-glycan type quantitative (glyco-TQ) method was first used to determine 37 the N-glycan type composition of immunoglobulin G1 (IgG1) Fc fragment. Then we applied the method 38 to analyze the glycan type profile of proteins in the breast cancer cell line MCF7, and quantitatively 39 revealed the N-glycan type micro-heterogeneity at both the glycopeptide and glycoprotein levels. The 40 novel quantitative strategy to evaluate the relative intensity of the six states of N-glycan type 41 glycosylation on each site provides a new avenue to investigate function of glycoproteins in broad areas, 42 such as cancer biomarker research, pharmaceuticals characterization and anti-glycan vaccine 43 development. 44 45 46 47 3 48Glycosylation is one of the most common post-translational modifications (PTM) 1 . Glycans exhibit 49 vast structural microheterogeneity which is mainly generated by variable glycan structures at each of 50 their specific glycosylation sites. The N-linked glycans are generally attached to the Asn at Asn-X- 51Ser/Thr consensus sequence, where X is any amino acid other than Pro 2 . The biosynthesis of N-linked 52 glycoproteins is under a complex sequence of enzymatically catalyzed events, leading to a variety of 53 diverse N-glycan structures. The diverse N-glycan structures are generally classified into three types: 54 high mannose, hybrid, and complex type glycans, with all N-glycans sharing a common penta-saccharide 55 (GlcNAc2 Man3) core structure 3 . Although the structure of glycan is variable, evidence shows that the 56 mammalian glycans are remarkably well conserved in certain organisms, expressing a distinct array of 57 glycan profiles under defined conditions 4 . 58Mass spectrometry (MS) is a powerful platform to comprehensively analyze protein glycosylation. 59However, due to the low abundance of glycosylated peptides and the heterogeneity of glycan structures, 60 N-glycopeptide enrichment is required. Several enrichment methods have been reported, including 61 lectin 5 and hydrazide chemistry-based methods 6,7 , boronic acid enrichment 8 , hydrophilic interaction 62 liquid chromatography (HILIC) 9 and metabolic labeling 10,11 . In general, these strategies for detecting N-63 linked glycosylated sites require an additional de-glycosylation step by N-glycosid...