A reassessment of the electrophoretic mobility of high molecular weight glutenin subunits of wheat

The high molecular weight subunits of wheat glutenin (HMW-GS) are important for bread-making quality. Their composition is routinely identified by Tris-glycine SDS-PAGE after reduction of glutenin disulfide bonds. However, the relation between their molecular weight and, hence, their primary structure, and their mobility in Tris-glycine SDS-PAGE has proven to be ambiguous. We demonstrate a Bis-Tris SDS-PAGE procedure with a neutral, instead of alkaline, pH in the gel and running buffers. In this method commonly occurring HMW-GS from wheat migrated in the order 5 > 2 ≈ 3 > 1 > 6 ≈ 2* > 7 > 8 > 9 > 12 > 10, which is different from the order obtained in the Tris-glycine system. HMW-GS were identified by N-terminal sequencing after isolation with RP-HPLC. Protein sequences of HMW-GS were further confirmed by LC-MS/MS analyses of chymotryptic peptides after comparing the MS data to amino acid sequences in protein databases. The numbers of amino acids of HMW-GS reflected well the mobility order in Bis-Tris SDS-PAGE. The results indicate that Bis-Tris SDS-PAGE may not only be used to identify HMW-GS, but also to estimate the length of their polypeptide chain, as such avoiding previously observed anomalies in migration order.

► We investigated a SDS-PAGE procedure with a neutral operating pH to separate HMW-GS.
► Different cultivars and purified HMW-GS were used to determine the migration order.
► Sequences of HMW-GS were identified with LC-MS/MS of their chymotryptic peptides.
► The chain length of HMW-GS reflected well the mobility order in SDS-PAGE.
► SDS-PAGE at a neutral pH avoided previously observed anomalies in migration order.