Structural characterization of the N-glycosylation of individual soybean β-conglycinin subunits

• A mass spectrometry approach provided novel structural information on N-linked glycans of soy conglycinin subunits.
• Sequential reversed-phase and activated porous graphitized carbon micro-chromatography allowed glycan purification.
• Oligosaccharide structures were characterized at each of the three protein subunits by analysis of underivatized oligosaccharides.
• Glycosylation sites were assigned by comparative analysis of native and deglycosylated samples.
• These data can produce novel information to assess functional properties of soybean proteins.

Soybean (Glycine max) 7S β-conglycinin is a seed storage protein consisting of homo- and hetero-trimers of three subunits, namely α (∼67 kDa), α′ (∼71 kDa), and β (∼50 kDa), non-covalently associated. The N-glycans released from the whole β-conglycinin have been already characterized by 1H NMR some decades ago. Nevertheless, the actual glycosylation of the potential sites and the glycoforms of the individual subunits have not been specifically investigated so far. In this study, up-to-date chromatographic, electrophoretic and mass spectrometric strategies have been combined to achieve the structural characterization of the glycoforms of the three individual β-conglycinin subunits. Glycosylation sites were assigned by analyzing the tryptic glycopeptides of the isolated subunits. Underivatized N-glycans were purified with a two-step clean-up, consisting in sequential reversed-phase and activated porous graphitized carbon micro-chromatography, and profiled by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS).