Submitted to: Electrophoresis
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/15/1999
Publication Date: N/A
Citation: Interpretive Summary: Worldwide, and in the U.S., barley is an important crop. In the U.S., the majority of the barley used for food is malted, with the brewing industry using most of the malt. Many of the characteristics of malt are determined by the proteins that are present in the barley from which it is made. To alter (improve) these characteristics, it is therefore necessary to understand which proteins are present in barley and what their biochemical characteristics are. One of the newest and best methods for studying grain proteins is called high performance capillary electrophoresis (HPCE). For this study, we used HPCE to separate and study the major barley proteins (called hordeins). We were able to separate them in less than 20 minutes and were able to use the resulting separation patterns to identify otherwise identical barley cultivars. The extraction and HPCE separation protocols described are fast, simple, and reliable. The hordeins were all separated in 16 minutes, and samples could be injected every 48 minutes. This method shows that HPCE is a vital method for characterizing hordein proteins and differentiating barley cultivars. This technique will be useful for researchers studying barley malting quality and for anyone who has to reliably identify morphologically identical barley varieties.
Technical Abstract: Extraction conditions, separation conditions, and capillary rinsing protocols were optimized for the separation of barley hordeins by free zone capillary electrophoresis. Stable hordein extracts were obtained with a single 5 minute extraction after the albumins and globulins were removed. Hordeins had to be reduced for optimal resolution. Optimum separation conditions for hordeins separations were 100 mM phosphate- glycine buffer containing 20% acetonitrile and 0.05% hydroxypropylmethyl- cellulose. The addition of zwitterionic sulfobetaine detergents containing hydrocarbon tails of eight and ten carbons slightly improved the resolution of the separations, but not enough to warrant their use on a routine basis. The migration positions of the hordein subclasses were determined by two dimensional reversed phase-high performance liquid chromatography x free zone capillary electrophoresis mapping. The hordein subclasses formed clusters similar to those of wheat gliadins. Separation -to-separation repeatability was good, with migration time relative standard deviations <1% for a 15 run period. For routine discrimination of cultivars, a 2 minute post separation rinse with 500 mM acetic acid was necessary to prevent protein build up on the capillary walls. An example of successfully differentiating barley cultivars using this technique is shown.