Differentiation of wheat high-molecular-weight glutenin subunits by targeted LC–MS/MS

High-molecular-weight glutenin subunits (HMW-GS) play an essential role in the end-use quality of wheat (Triticum aestivum L.). We developed a targeted liquid chromatography-tandem mass spectrometry method in parallel reaction monitoring (LC–MS/MS-PRM) to detect and differentiate wheat HMW-GS in German wheat cultivars with known (37 cultivars) and unknown (58 cultivars) composition. The newly developed method is suitable to unambiguously identify Ax1, Ax2*, Bx6, Bx14, Dx2, Dx5, Dy10, Dy12, as well as any absence of Ax, but cannot distinguish Bx7 and Bx17 and identify the variant of By due to high sequence identity to Dy and within By. The method is further suited to clearly conclude, if the sample is a mixture of at least two cultivars or consists of only one cultivar. In comparison to gel-based methods (SDS-PAGE), UV-detection after LC (RP-HPLC–UV) and MS of intact proteins (MALDI-TOF–MS), LC–MS/MS has a high resolution, is less biased by interpretation and provides more insights on molecular level. The used procedure can be applied to expand the LC–MS/MS-PRM method for more HMW-GS or even to other wheat proteins, e.g., low-molecular-weight glutenin subunits (LMW-GS), in future. This study describes the first MS-based method on peptide level for the differentiation of wheat HMW-GS.