Improvements in Cereal Protein Separations by Capillary Electrophoresis : Resolution and Reproducibility 1

Cereal Chem. 73(1):81-87 Capillary electrophoresis rinsing protocols and buffer compositions 0.05% hydroxypropylmethylcellulose. Twenty percent acetonitrile prowere optimized to improve the resolution and reproducibility of cereal vided optimal resolution, while maintaining excellent reproducibility. prolamin separations. Capillary cleaning protocols were varied to imThat buffer provided high resolution of wheat and oat prolamins. Resoprove reproducibility. Four cleaning regimes were tested; the optimum lution of rice prolamins also improved using that buffer, but best resoluwas a 4-min rinse with IM phosphoric acid. Several buffer modifiers tion of rice prolamins was found when lauryl-sulfobetain at its critical were tested for use with 100 m.M phosphate buffer (pH 2.5) containing micelle concentration (26 mM) was also added. Cereal proteins have been studied by many analytical techniques due to their relationships with quality and varietal identification. Polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography are currently the methods of choice. Gel electrophoresis, acid-PAGE, and sodium dodecyl sulfate-PAGE are the established methods for protein separations. However, these methods have several drawbacks, including the use of toxic reagents, long analysis times, and difficulties in quantifying and interpreting data. Electrophoretic methods and extraction conditions used to differentiate cultivars of all major cereal crops were recently reviewed by Lookhart (1990) and Lookhart and Wrigley (1995). Reversed-phase high-performance liquid chromatography has also been used to differentiate cereal cultivars, alone and as a complement to acid-PAGE, for rice (Lookhart et al 1987, 1991; Huebner et al 1991; Lookhart and Juliano 1994), oats (Lookhart 1985, Lookhart and Pomeranz 1985, Bakhella et al 1992), barley (Marchylo and Laberge 1980, Heisel et al 1986), and wheat (Bietz 1983, Lookhart et al 1986, Lookhart and Albers 1988, Bakhella et al 1991, Huebener and Bietz 1994). Recently, wheat proteins were characterized by a new technique, capillary electrophoresis (CE) (Bietz and Lookhart 1994; Werner et al 1994; Lookhart et al 1994, Bietz and Schmalzried 1995; Lookhart and Bean 1995a,b). An aluminum lactate buffer, in conjunction with capillaries coated with Microcoat (PerkinElmer), was used by Weiner et al (1994) to separate gliadins. The other authors (Bietz and Lookhart 1994; Bietz and Schmalzried 1995; Lookhart and Bean 1995a,b) used primarily a low-pH (2.5) commercial (Bio-Rad) phosphate buffer to separate cereal proteins. Further modifications of this method reduced the time lCooperative investigations, U.S. Department of Agriculture, Agricultural Research Service and the Department of Grain Science and Industry, Kansas State University. Contribution 95-534-J, Department of Grain Science and Industry, Kansas State Agricultural Experiment Station, Manhattan, KS 66506. Mention of firm names or trade products does not constitute endorsement by the U.S. Department of Agriculture over others not mentioned. Research Chemist, USDA-ARS, Grain Marketing Research Laboratory, Manhattan, KS 66502. Fax 913/776-2792. Research Assistant, Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506. U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Publication no. C-1996-01 1 1-01 R. C 1996 American Association of Cereal Chemists, Inc. and optimized the resolution and reproducibility for separation of those proteins (Lookhart and Bean 1995a). Gliadins were separated in less than 10 min, with a relative standard deviation (RSD) of 0.6% for migration times. CE of cereal proteins has recently undergone considerable development. It is a rapid technique, easily automated, that requires little sample or reagents (Chen 1991). We have shown that resolution and reproducibility could be further improved when wash procedures were optimized and buffers containing organic modifiers were used. These studies confirm that CE is a highly resolving, reproducible technique for rapid cereal cultivar identification and protein characterization. MATERIALS AND METHODS