Rapid separation and identification of wheat HMW glutenin subunits by UPLC and comparative analysis with HPLC

High molecular weight glutenin subunits (HMW-GS) are among the major determinant on wheat bread-making quality. Rapid ultra-performance liquid chromatography (UPLC) methods with high resolution and reproducibility for HMW-GS separation were established in this work. A sample could be completely analyzed in less than 12 min by using the optimized UPLC conditions: gradually increasing elution gradient from 21% to 47% in 30 min at flow rate 0.55 ml/min and separation temperature 55C. The results from 15 consecutive runs showed that UPLC patterns of HMW-GS including peak migration time, height and peak area displayed high reproducibility. In addition, the analysis consumes small amounts of reagent. A total of 34 subunits from 111 wheat cultivars and gene bank accessions were well separated and showed clearly different UPLC patterns. Thus all subunits could be readily identified, especially for 1Dx and 1By subunits that were not separable by traditional HPLC. The results from different growing environments demonstrated that the subunit elution times were highly stable, displaying significant changes in the subunit peak heights and areas, reflecting their expression amounts. In comparison with the state-of-the-art HPLC method, UPLC has obvious advantages in separation time, resolution and reagent consuming, which provides an alternative powerful technique for wheat storage protein studies and quality improvement.