Separation of Wheat Proteins by Two‐Dimensional Reversed‐Phase High‐Performance Liquid Chromatography Plus Free Zone Capillary Electrophoresis

Gliadins and glutenins from four hard red winter wheat cultivars were separated by a novel two‐dimensional (2D) technique. Protein extracts were separated by reversed‐phase high performance liquid chromatography as the first dimension with each 30‐sec interval collected separately. Those fractions were then separated by free‐zone capillary electrophoresis (FZCE) for the second dimension. Data was combined into 2D surface contour plots similar to traditional gel electrophoresis 2D maps. For HPLC, C 8 and C 18 columns were used in the first dimension to separate gliadins and glutenins, respectively. Uncoated fused silica capillaries (27 cm × 25 μm, i.d.) were used for the 2D FZCE separations. Differences in the 2D maps of both gliadin and glutenin fractions were found between pairs of both closely related and sister lines that varied in quality.