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.

A Dionex Ultimate 3000 UHPLC system (ThermoFisher Scientific, Waltham, MA, USA) was coupled to a Q Exactive Plus Orbitrap mass spectrometer (ThermoFisher Scientific). The digests were dissolved in 100 µL water containing acetonitrile (2%) and formic acid (0.1%) and separated by an Acquity UPLC® column (HSS T3, 1.8 μm, 2.1 × 150 mm, Waters, Milford, MA, USA). The injection volume was 10 µL, the flow rate was 0.2 mL/min, solvent A was water (0.1% formic acid), solvent B was acetonitrile (0.1% formic acid), the gradient was 0–1 min 5–8% B, 1–14 min 8–18% B, 15–18 min 18–25% B, 18–23 min 25–40% B, 23–25 min 40–80% B, 25–27 min 80% B, 27–28 min 80–5% B, 28–35 min 5% B and the column temperature was 55 °C. The eluate from the analytical column was sprayed via a ESI source (ThermoFisher Scientific) into the MS at a source voltage of 3.5 kV, at a capillary temperature of 290 °C and S‐lens level of 50 (sheath gas flow rate: 40, aux gas flow rate: 10, sweep gas flow rate 1, aux gas heater temperature: 100 °C). The Q Exactive Plus was set to PRM modus with a resolution of 17,500, injection time of 50 ms, an automatic gain control of 2e4, an isolation width of 1.6 m/z and a normalized collision energy of 18, 23 and 27 for fragmentation.

In total, 37 common wheat samples with known HMW-GS composition and 58 ones with unknown composition that were grown and harvested in different years and locations were used. The kernels were milled with different mills and most of them were white flour, but there were wholemeal flours as well. The flours were stored for several weeks, but not longer than for three years. Flour (100 mg) was extracted two times with a 50/50 (v/v) mixture of propan-1-ol and water containing 1% dithiothreitol (w/v) at 60 °C for 30 min. After centrifugation, the volume of the combined supernatants was adjusted to be 2 mL with the extraction solution. Propan-1-ol (0.5 mL) was added to obtain a propan-1-ol concentration of 60%. After incubation and centrifugation, the residue containing the HMW-GS was directly used for total digestion. The residue containing the isolated HMW-GS was dissolved in a 1:1 mixture of propan-1-ol and Tris-HCl. Reduction was performed using TCEP and alkylation using CAA. After concentration in a rotational vacuum concentrator, the proteins were digested with a mixture of trypsin and chymotrypsin overnight (enzyme-substrate ratio: 1:20). The digestion was stopped with trifluoroacetic acid and the peptides were purified by solid-phase extraction (Discovery®, DSC-18, 100 mg volume). The peptides were eluted with water/acetonitrile containing 0.1% formic acid concentrated in a rotational vacuum concentrator and stored at −20 °C prior to analysis.