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

Soybean (Glycine max) 7S β-conglycinin is a seed storage protein consisting of homo- and hetero-trimers of three subunits, namely α (~67kDa), α' (~71kDa), and β (~50kDa), 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).