Electrophoresis-based analysis of wheat seed proteins affecting flour quality

Abstract

Glutenin and gliadin, the major storage proteins in wheat seeds, significantly affect wheat flour’s secondary processing properties. These proteins absorb water and form a network of protein complexes called gluten. Bread wheat is a hexaploid species composed of three different genomes, A, B, and D, each containing homoeologous genes encoding proteins with similar functions. This results in the production of multiple isoforms with similar functions but slightly different sequences. In addition, some of the gluten proteins undergo post-translational modifications. Another characteristic of glutenin and gliadin is that they are rich in glutamine and have many repetitive sequences. These characteristics were why protein identification by protein shotgun analysis using only mass spectrometry is difficult.

We have succeeded in identifying a large number of proteoforms (isoforms+post-translationally modified proteins) by using two-dimensional electrophoresis combined with mass spectrometry, and we found that specific proteoforms correlated with the Fb value of the alveograph, which indicates the quality of the flour. On the other hand, disulfide bonds in gluten have been considered to be closely related to the quality. However, details of the disulfide bonding patterns have not been determined due to the difficulty of analysis of large gluten molecules. Therefore, we successfully fragmented glutenin with cyanogen bromide and determined the disulfide bonding patterns using diagonal gel electrophoresis and mass spectrometry.