Improving high-performance capillary electrophoresis methods for characterizing the proteins of wheat, barley, oats, rice, maize, and sorghum: 'Putting HPCE to work'

Food proteins play important roles in food functionality, nutrition, and human health. One important class of food proteins are those found in cereal grains. Cereal proteins are unique in many ways: they are highly complex and heterogeneous, are often difficult to extract, and aggregate readily, making them difficult to characterize. Because of the economic importance and widespread use of cereal proteins, however, many techniques have been used for their analysis. High-performance capillary electrophoresis (HPCE) is one of the newest techniques to be so used. HPCE offers several benefits to the study of cereal proteins. HPCE is fully automated, allowing for unattended operation and produces digitized data that is easily stored, analyzed, and quantified. This is often difficult to do with traditional electrophoresis data. Furthermore, HPCE produces little toxic waste and is inexpensive to operate on a day-to-day basis. Considerable research has been done on developing methods to separate cereal proteins by HPCE. The focus of this dissertation will be on improving existing methods for separating cereal proteins with emphasis on making HPCE a rapid, reliable, and rugged tool for cereal chemists to use. The first part of this research describes the use of low conductive and isoelectric buffers to produce very rapid (2–10 min), high resolution, reproducible separations. To achieve this, two specialized buffer systems based on phosphate-glycine and iminodiacetic acid buffers were developed. These buffers were used to separate storage proteins from wheat, oats, rice, barley, and rye. Slight modifications of the phosphate glycine buffer was used to achieve successful separations of maize and sorghum storage proteins. For this work, 60% acetonitrile was added to the buffer to help maintain protein solubility. In the second part, methods for separating cereal proteins by SDS-CE are described using polymers in dynamically coated capillaries. Finally, improved methods for sample preparation are reported. It was found that pre-extraction of albumins and globulins resulted in better reproducibility and less stringent capillary rinsing proteins. The factors involved in achieving good quantitative reproducibility are reported in the final part. Critical factors found were making a clean, straight cut on the capillary ends, humidifying the sample vials, and optimizing the injection conditions. Decreasing the initial voltage ramp up time was found to increase the efficiency of the separations.