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 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.
Department of Environmental Health Sciences, IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.
Silvia
S
Schiarea
Lolita
L
Arnoldi
Roberto
R
Fanelli
Eric
E
De Combarieu
Chiara
C
Chiabrando
D000595:Amino Acid Sequence; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D021122:Protein Subunits; D020543:Proteome; D040901:Proteomics
D006023:Glycoproteins; D010940:Plant Proteins; D011134:Polysaccharides; D021122:Protein Subunits; D020543:Proteome