Characterization of seed hordeins and varietal identification in three barley species by high-performance capillary electrophoresis

Separation and characterization of the seed horde ins in three barley species (Hordeum vulgare L., 2n=2x= 14, Hordeum spontaneum L., 2n=2x= l4 and Hordeum bulbosum L., 2n=4x=28) by high-performance capillary electrophoresis (HPCE) and the potential use of HPCE for varietal identification were investigated. Different protein extraction and electrophoresis conditions were tested and the following method appeared to be optimal for seed hordein analysis of barley: hordeins were extracted with 40% aqueous ethanol (v/v) and separated in 0.1 M phosphate-glycine buffer (pH 2.5), containing 20% acetonitrile (ACN) and 0.05% hydroxypropylmethyl-cellulose (HPMC), using a 27 cm (25 μm ID, 20 cm L0) capillary at 12.5 kV and 40 °C on a BioFocus 3000 HPCE instrument (Bio-Rad). Reproducibility for run-to-run separations was good by rinsing the capillary with I M phosphoric acid and separation buffer for 2 min, respectively, after each analysis. The high-resolution and rapid separation and characterization for barley hordeins were achieved with the above method, and two protein subclasses, B and C hordeins could be readily separated and identified. Hordein HPCE patterns of three barley species showed apparent qualitative and quantitative differences. In particular, the two diploid species showed relatively similar hordein patterns, while the tetraploid specie exhibited evident differences in protein components and elution time, especially in the C hordein range. HPCE appears to be capable of rapid, high-resolution, reproducible, and automated separation of seed hordeins, and therefore, it has a great potential for protein separation, characterization and varietal identification in barley breeding and end-use.