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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: Schiarea, Silvia, Arnoldi, Lolita, Fanelli, Roberto, De Combarieu, Eric, Chiabrando, Chiara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771881/
https://www.ncbi.nlm.nih.gov/pubmed/24069245
http://dx.doi.org/10.1371/journal.pone.0073906
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author Schiarea, Silvia
Arnoldi, Lolita
Fanelli, Roberto
De Combarieu, Eric
Chiabrando, Chiara
author_facet Schiarea, Silvia
Arnoldi, Lolita
Fanelli, Roberto
De Combarieu, Eric
Chiabrando, Chiara
author_sort Schiarea, Silvia
collection PubMed
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 Man(2)(Xyl) (Fuc) GlcNAc(2), Man(3)(Xyl) (Fuc) GlcNAc(2), GlcNAcMan(3)(Xyl) (Fuc) GlcNAc(2) and GlcNAc (2)Man(3)(Xyl) (Fuc) GlcNAc(2). 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 Asn(131), 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.
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spelling pubmed-37718812013-09-25 In-Depth Glycoproteomic Characterization of γ-Conglutin by High-Resolution Accurate Mass Spectrometry Schiarea, Silvia Arnoldi, Lolita Fanelli, Roberto De Combarieu, Eric Chiabrando, Chiara PLoS One Research Article 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 Man(2)(Xyl) (Fuc) GlcNAc(2), Man(3)(Xyl) (Fuc) GlcNAc(2), GlcNAcMan(3)(Xyl) (Fuc) GlcNAc(2) and GlcNAc (2)Man(3)(Xyl) (Fuc) GlcNAc(2). 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 Asn(131), 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. Public Library of Science 2013-09-12 /pmc/articles/PMC3771881/ /pubmed/24069245 http://dx.doi.org/10.1371/journal.pone.0073906 Text en © 2013 Schiarea et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Schiarea, Silvia
Arnoldi, Lolita
Fanelli, Roberto
De Combarieu, Eric
Chiabrando, Chiara
In-Depth Glycoproteomic Characterization of γ-Conglutin by High-Resolution Accurate Mass Spectrometry
title 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_short 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771881/
https://www.ncbi.nlm.nih.gov/pubmed/24069245
http://dx.doi.org/10.1371/journal.pone.0073906
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