Mass spectrometry of in-gel digests reveals differences in amino acid sequences of high-molecular-weight glutenin subunits in spelt and emmer compared to common wheat
High-molecular-weight glutenin subunits (HMW-GS) play an important role for the baking quality of wheat. The ancient wheats emmer and spelt differ in their HMW-GS pattern compared to modern common wheat and this might be one reason for their comparatively poor baking quality. The aim of this study was to elucidate similarities and differences in the amino acid sequences of two 1Bx HMW-GS of common wheat, spelt and emmer. First, the sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) system was optimized to separate common wheat, spelt and emmer Bx6 and Bx7 from other HMW-GS (e.g., 1Ax and 1By) in high concentrations. The in-gel digests of the Bx6 and Bx7 bands were analyzed by untargeted LC-MS/MS experiments revealing different UniProtKB accessions in spelt and emmer compared to common wheat. The HMW-GS Bx6 and Bx7, respectively, of emmer and spelt showed differences in the amino acid sequences compared to those of common wheat. The identities of the peptide variations were confirmed by targeted LC-MS/MS. These peptides can be used to differentiate between Bx6 and Bx7 of spelt and emmer and Bx6 and Bx7 of common wheat. The findings should help to increase the reliability and curation status of wheat protein databases and to understand the effects of protein structure on the functional properties.
Flour (100 mg) was extracted two times with 50 % propan-1-ol (1 mL, v/v) containing 1% DTT (w/v) at 60 °C for 30 min. After centrifugation for 25 min at 22 °C and 3,750 g, the supernatants were combined in conical centrifuge tubes and 50% propan-1-ol was added to obtain a defined total volume of 2 mL. The concentration of propan-1-ol was increased to 60% by adding propan-1-ol (0.5 mL). The samples were incubated at 22 °C for 30 min and then, centrifuged at 22 °C and 3,750 g for 25 min. The supernatant was discarded and the residue containing the HMW-GS was directly used either for SDS-PAGE or for total digestion. The precipitated HMW-GS of 100 mg flour were dissolved in propan-1-ol (320 µL) and Tris-HCl (320 µL, 0.5 mol/L, pH 8.5). Reduction was carried out with TCEP (50 µL, 0.05 mol/L TCEP in 0.5 mol/L Tris-HCl, pH 8.5) for 10 min at 60 °C and alkylation with CAA (100 µL, 0.5 mol/L CAA in 0.5 mol/L Tris-HCl, pH 8.5) for 45 min at 37 °C in the dark. The solutions were concentrated in a rotational vacuum concentrator for 3 h at 30 °C. Then, the proteins were either hydrolyzed with chymotrypsin (1 mL, 50 µg/mL in Tris-HCl, 0.1 mol/L, pH 7.8) or with a mixture of trypsin and chymotrypsin (1 mL, 25 µg/mL each in Tris-HCl, 0.1 mol/L, pH 7.8) at 37 °C overnight. The digestion was stopped with TFA and the peptides were purified by solid-phase extraction (SPE, Discovery®, DSC-18, 100 mg volume, Sigma). The Peptide were eluted with 40 % ACN (1 mL, v/v, 0.1 % FA), concentrated in a rotational vacuum concentrator and stored at -20 °C prior to analysis.
Total HMW-GS digests were dissolved in water/ACN (500 µL, 98/2, v/v, 0.1% FA). An ExionLC (Sciex, Darmstadt, Germany) was connected to a 6500+ QTrap (Sciex). The peptides (injection volume 10 µL) were separated on a bioZen PS-C18 Peptide column (150×2.1 mm, 1.6 µm, Phenomenex, Aschaffenburg, Germany) using a flow rate of 0.3 mL/min, solvent A, 1% FA in water, solvent B, 1% FA in ACN, a gradient of 0–0.5 min 5% B, 0.5–14 min 5–40% B, 14–15 min 40–100% B, 15–16 min 100% B, 16–17 min 100–5% B, 17–20 min 5% B and a column temperature of 40 °C. The MS was operated in positive ESI mode. Ion spray voltage was set at 5500 eV and source temperature at 550 °C. Nitrogen served as nebulizing gas at 0.55 MPa, as heating gas at 0.65 MPa and as curtain gas at 0.35 MPa. Using nitrogen in the collision cell, peptide fragmentation was performed by using calculated peptide specific collision energies in Skyline (version 4.2, MacCoss Lab Software, University of Washington, Seattle, WA, U.S.A). Instrument control and data acquisition were performed with Sciex Analyst software (version 1.6.3).
The common wheat cultivars Oxal and Sonett were kindly provided by Lorenz Hartl (Bayerische Landesanstalt fuer Landwirtschaft, LfL, Freising, Germany) and Ares, Dekan, Format, Kanzler and Ritmo by Andreas Börner (Leibniz Institute of Plant Genetics and Crop Plant Research, Resources Genetics and Reproduction, Gatersleben, Germany). All other common wheat, spelt and emmer cultivars were kindly provided by Friedrich Longin (University of Hohenheim, LSA-Research Group Wheat, Stuttgart, Germany). The kernels were milled to white flour using a Quadrumat Junior Mill (Brabender, Duisburg, Germany) and the flours were sieved (mesh size 0.2 mm).