In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.

The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergeni...

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Autores principales:	Silvia Schiarea, Lolita Arnoldi, Roberto Fanelli, Eric De Combarieu, Chiara Chiabrando
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/15fa770047894ee38c4b2e76aecefb5d
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spelling	oai:doaj.org-article:15fa770047894ee38c4b2e76aecefb5d2021-11-18T08:55:32ZIn-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.1932-620310.1371/journal.pone.0073906https://doaj.org/article/15fa770047894ee38c4b2e76aecefb5d2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24069245/?tool=EBIhttps://doaj.org/toc/1932-6203The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography-multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl) (Fuc) GlcNAc2, Man3(Xyl) (Fuc) GlcNAc2, GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2. These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.Silvia SchiareaLolita ArnoldiRoberto FanelliEric De CombarieuChiara ChiabrandoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e73906 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Silvia Schiarea Lolita Arnoldi Roberto Fanelli Eric De Combarieu Chiara Chiabrando In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
description	The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography-multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl) (Fuc) GlcNAc2, Man3(Xyl) (Fuc) GlcNAc2, GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2. These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.
format	article
author	Silvia Schiarea Lolita Arnoldi Roberto Fanelli Eric De Combarieu Chiara Chiabrando
author_facet	Silvia Schiarea Lolita Arnoldi Roberto Fanelli Eric De Combarieu Chiara Chiabrando
author_sort	Silvia Schiarea
title	In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
title_short	In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
title_full	In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
title_fullStr	In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
title_full_unstemmed	In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
title_sort	in-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/15fa770047894ee38c4b2e76aecefb5d
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In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.

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