Differentiation of wheat high-molecular-weight glutenin subunits

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.

高分子量谷蛋白亚基（High-molecular-weight glutenin subunits, HMW-GS）对普通小麦（Triticum aestivum L.）的最终加工品质具有至关重要的作用。本研究建立了平行反应监测模式靶向液相色谱-串联质谱法（parallel reaction monitoring-liquid chromatography-tandem mass spectrometry, LC-MS/MS-PRM），用于检测和区分已知组成（37个品种）与未知组成（58个品种）的德国小麦品种中的小麦HMW-GS。该新开发方法可精准鉴定Ax1、Ax2*、Bx6、Bx14、Dx2、Dx5、Dy10、Dy12以及所有Ax亚基缺失的样本，但由于By亚基与Dy亚基及自身各亚型间序列同源性较高，其无法区分Bx7与Bx17，也无法鉴定By亚基的变异型。此外，该方法可明确判断样本是否为至少两个小麦品种的混合样，抑或仅来自单一品种。相较于基于凝胶的检测方法（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，SDS-PAGE）、液相色谱联用紫外检测法（反相高效液相色谱-紫外检测法，RP-HPLC-UV）以及完整蛋白质质谱分析法（基质辅助激光解吸电离飞行时间质谱，MALDI-TOF-MS），液相色谱-串联质谱（LC-MS/MS）法具有更高分辨率，结果解读偏倚更低，且可在分子层面提供更丰富的信息。未来，本研究采用的实验流程可用于拓展LC-MS/MS-PRM法的检测范围，使其能够鉴定更多HMW-GS，甚至可应用于其他小麦蛋白质，例如低分子量谷蛋白亚基（low-molecular-weight glutenin subunits, LMW-GS）。本研究首次报道了基于肽段层面的质谱分析法，用于区分小麦HMW-GS。