In-Depth Glycoproteomic Characterization of γ-Conglutin by High-Resolution Accurate Mass Spectrometry

Silvia Schiarea, Lolita Arnoldi, Roberto Fanelli, Eric De Combarieu, Chiara Chiabrando

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article numbere73906
JournalPLoS One
Volume8
Issue number9
DOIs
Publication statusPublished - Sep 12 2013

Fingerprint

Glycosylation
Mass spectrometry
Polysaccharides
Mass Spectrometry
Fucose
fucose
polysaccharides
mass spectrometry
glycosylation
amino acid sequences
Health
glycopeptides
Lupinus
health promotion
Proteins
Glycopeptides
Xylose
Liquid chromatography
xylose
Liquid Chromatography

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

In-Depth Glycoproteomic Characterization of γ-Conglutin by High-Resolution Accurate Mass Spectrometry. / Schiarea, Silvia; Arnoldi, Lolita; Fanelli, Roberto; De Combarieu, Eric; Chiabrando, Chiara.

In: PLoS One, Vol. 8, No. 9, e73906, 12.09.2013.

Research output: Contribution to journalArticle

Schiarea, Silvia ; Arnoldi, Lolita ; Fanelli, Roberto ; De Combarieu, Eric ; Chiabrando, Chiara. / In-Depth Glycoproteomic Characterization of γ-Conglutin by High-Resolution Accurate Mass Spectrometry. In: PLoS One. 2013 ; Vol. 8, No. 9.
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