Mass Spectrometric Analysis of N-Glycosylation Sites and N-Glycans of β-Conglycinin†

doi:10.7503/cjcu20170584

Abstract

Abstract:

As one of common allergenic food, soybean makes people allergic attributed to it’s allergen β-conglycinin, of which the analysis of glycosylation sites and N-glycan structure has a significant meaning for understanding antigenic properties and sensitization mechanism of soybean in-depth. However, due to the limitations of analytical techniques, the corresponding relationship between glycosylation sites and N-glycan structure is lack of comprehensive understanding. In this study, β-conglycinin was hydrolyzed into peptide fragments using Trypsin and Pepsin, followed by enrichment of glycopeptides using Con A affinity chromatography. Subsequently, two aliquots of the glycopeptide samples were subjected to digestion with PNGase F and Endo H, respectively. The recovered peptides and glycans were analyzed in detail by means of electrospray ionization mass spectrometry(ESI-MS) and tandem mass spectrometry(MS/MS). The obtained data of peptides were retrieved using the Mascot software. These results show that β-conglycinin has 5 N-glycosylation sites, including the 199th and 455th Asn residues of the α subunit, the 215th and 489th Asn residues of the α' subunit and the 326th Asn residue of the β subunit, and each glycosylation site was modified by the four high-mannose type N-glycans, including H5N2, H6N2, H7N2 and H8N2. This study provides a new strategy for the identification of glycosylation sites and corresponding glycan structures of various glycoproteins and offers a structural basis for deep understanding of the specificity of soybean protein glycoprotein epitopes and allergic mechanism.

Key words: β-Conglycinin, Glycosylation site, N-Glycan, Mass spectrometry

TrendMD:

LI Lingmei, WANG Chengjian, HAN Jianli, LIU Rendan, WEN Yanan, WEI Ming, JIN Wanjun, HUANG Linjuan, WANG Zhongfu. Mass Spectrometric Analysis of N-Glycosylation Sites and N-Glycans of β-Conglycinin^†[J]. Chem. J. Chinese Universities, 2018, 39(3): 427.

Figures/Tables 7

Fig.1 SDS-PAGE profile of β-conglycinin

Fig.2 ESI-MS spectrum of the glycopeptides of β-conglycinin purified using Con A Structural formulas: ■ : N-acetylglucosamine; ●: mannose.

Fig.3 Mass spectra of the peptides obtained via digestion by PNGase F(A) and Endo H(B)

Fig.4 Sequential identification of the peptide PVVVN#ATSNL digested from PNGase F(A) and Endo H(B) by MS/MS

Table 1 Summary of the glycosylation sites of β-conglycinin

Subunit	Glycopeptide sequences	Site position	Subunit	Glycopeptide sequences	Site position
α' subunit	ILN#GTA	215	α subunit	PVVVN#ATSNL
	VILN#GTA			ILN#GTA	199
	IVILN#GTA			VILN#GTA
	LIVILN#GTA			IVILN#GTA
	PVMVN#A	489		LIVILN#GTA
α subunit	PVVVN#A	455	β subunit	VVN#ATSNL	326
	PVVVN#ATSN

Fig.5 ESI-MS spectra of the N-glycans obtained by hydrolysis using PNGase F(A) and Endo H(B)

Fig.6 Summary of glycosylation sites and corresponding N-glycan structures of β-conglycinin

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