In-Depth Glycoproteomic Characterization Of Gamma-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 gamma-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 gamma-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 beta 1,2-xylose and core alpha 1-3-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 gamma-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.