Accelerating wheat (Triticum aestivum) proteomics using a tandem digestion approach

Proteomics research on wheat grain can provide valuable information regarding nutritional quality, stress acclimation and potential impacts on human health. Trypsin has traditionally served as the benchmark protease in wheat proteomics research. Nevertheless, trypsin encounters some limitations, primarily due to the scarcity of lysine and arginine cleavage sites in this proteome. In this research, a tandem digestion strategy was proposed to enhance the wheat proteome analysis. By combining the complementary cleavage specificities of glutamyl endoproteinase (Glu-C) and trypsin, our method significantly improved the identification of both the gluten proteins and specific metabolic proteins. Additionally, we observed an increase in the numbers of immunoreactive proteins identified, which are known to trigger severe allergic reactions in certain individuals. Compared with sole trypsin digestion, the tandem digestion approach enabled access to regions of the wheat proteome that are inaccessible to trypsin alone. Consequently, our tandem digestion strategy significantly expanded the depth of wheat proteome analysis, serving as a valuable complement to the conventional trypsin-based approach.