Fast separation and characterization of water-soluble proteins in wheat grains by reversed-phase ultra performance liquid chromatography (RP-UPLC)

Water-soluble proteins account for about 10% of total grain proteins in wheat (Triticum aestivum L.) and have specific functions in plant growth and development. In this study, a reversed-phase ultra performance liquid chromatography (RP-UPLC) method was trialed and the experimental conditions were optimized for rapidly separating and characterizing water-soluble proteins in wheat grains, and a comparative analysis with traditional RP-HPLC was performed. Under optimized separation conditions, fast, high resolution and reproducible RP-UPLC separation for water-soluble proteins could be obtained by gradually increasing eluting gradient from 21% to 47% in 30 min at 0.6 ml/min and 60 °C. Using this method, separation and characterization of water-soluble protein in wheat grains could be completed in less than 20 min for one sample, and the resolution and efficiency were significantly higher than those obtained with RP-HPLC. In addition, RP-UPLC consumed smaller amounts of samples and reagents as well. The optimized RP-UPLC could be used as an effective and alternative method for rapid separation and characterization of water-soluble proteins in wheat cultivar and germplasm evaluation, genetic and biochemical studies on grain proteins, and environmental influence analysis of water-soluble proteins.