Separation of cereal proteins by reversed-phase high-performance liquid chromatography

Cereal proteins have been extremely difficult to purify and characterize owing to their heterogeneity, poor solubility and tendency to polymerize. High-performance liquid chromatography (HPLC) on a 300 A reversed-phase (RP)(C18) support (Syn Chropak RP-P), using acetonitrile as organic modifier in the presence of trifluoroacetic acid, has been found to be capable of high-resolution separations of these proteins; the resolution is often better than that obtained by any other chromatographic or electrophoretic method. Examples are presented showing separations of low-molecular-weight gliadins, omega-gliadins and ethanol-soluble reduced glutenin subunits from wheat and of zein from corn. In addition, proteins may be directly extracted from ground single kernels and subsequently analyzed by RP-HPLC; applications in genetic studies, in breeding programs and in varietal identification are proposed. In addition to its high resolution, RP-HPLC is superior to most other methods in speed, sensitivity, reproducibility and suitability for quantitation. Polypeptide chains of molecular weight up to 133,000 are recovered in high yields and the column capacity is high, demonstrating that RP-HPLC is suitable for both preparative and analytical separations of proteins. RP-HPLC resolves proteins primarily on the basis of differences in surface hydrophobicity, so it therefore complements, rather than duplicates, other techniques that separate proteins on the basis of size or charge. RP-HPLC promises to become an invaluable technique for the fractionation and characterization of proteins from cereals and other sources.