Structural analysis of wheat glutenins by diagonal electrophoresis for disulfide bond detection

Gluten is a wheat-specific protein aggregate that strongly affects dough properties and mainly comprises glutenins and gliadins. Glutenins form large complexes through intermolecular disulfide bonds, which have not been fully structurally characterized. Herein, the structures of glutenins in four wheat cultivars were analyzed via CNBr digestion, which resulted in cleavage at methionine residues to afford peptides with molecular weights close to those of intact high-molecular-weight glutenin subunits (HMW-GSs). The diagonal electrophoresis of these peptides enabled the partial visualization of the disulfide bond–connected glutenin structure. This structure was formed before the mixing of flour and water and was present in all cultivars, with its composition depending on the cultivar-specific glutenin genotype. The constituent proteins were identified as HMW-GSs, low-molecular-weight glutenin subunits, α-gliadin, and serpin using liquid chromatography-tandem mass spectrometry. These results demonstrated that HMW-GSs formed a complex with disulfide bonds, even though the cysteines used for head-to-tail bonding were cleaved by CNBr. Until now, the HMW-GSs in glutenins were thought to be linearly disulfide-bonded via head-to-tail bonds. However, our results suggest that the cysteines in the central part are also disulfide-bonded, forming a branched structure.