Ultrafast Capillary Electrophoretic Analysis of Cereal Storage Proteins and Its Applications to Protein Characterization and Cultivar Differentiation

Abstract

Free zone capillary electrophoresis conditions have been improved to allow rapid (2-8 min) separations of grain proteins from several cereals (wheat, oats, rice, barley, and rye) with high resolution and reproducibility. This new method utilized the isoelectric compound iminodiacetic acid (IDA) in conjunction with 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. Cultivars of all cereals tested could be differentiated in 3 min, including wheat, using either prolamin or glutelin protein patterns. Resolution was similar to or higher than that of separations in other acidic buffers. Migration time repeatability was excellent with run-to-run variability <1% RSD, day-to-day <1.4% RSD, and capillary-to-capillary <3.3% RSD. Because larger inner diameter capillaries (50 microm) could be used with this buffer, sensitivity was improved and capillary rinse times could be reduced when compared to smaller capillaries (25 microm i.d.). This also served to reduce total separation time so that the majority of cereal storage protein from several types of cereals could be analyzed with total analysis times of 2-8 min with extremely high resolution and repeatability. This method would allow unattended, high-throughput ( approximately 180-400 samples/24 h) analysis of cereal proteins without the generation of much organic solvent waste as well as automated data analysis and storage.

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins

“…CE is capable of separating proteins by several different mechanisms, viz., size (SDS-capillary gel electrophoresis (SDS-CGE)), charge density (CZE), and pI (CIEF) [3]. Furthermore, CE is capable of providing high resolution, rapid, ultra-small sample volume, low solvent consumption, and automated separations of proteins, thus complementing HPLC [8].…”

“…In some instances it is sufficient to know the total amount of protein present. More often, however, it is desirable to fractionate the cereal proteins using high-resolution separation techniques, such as chromatography or electrophoresis [3].…”

Development of a capillary electrophoresis method for the determination of soybean proteins in soybean–rice gluten‐free dietary products

“…The separation media consisted of 80 mM phosphate buffer at different pH values (2.0, 2.5, and 7.0) and 80 mM borate buffer at pH 8.5, all of them modified with 20% v/v ACN. The addition of this organic modifier at this percentage was found to be adequate for the separation of cereal proteins by CE, particularly, for rice proteins [18,24,25]. All these first experiments were made using UV-absorption detection at 200 nm (peptide bond absorption) in order to avoid the missing of any peak that could be interesting for the purpose of this work.…”

Minor Wheat Protein Fractions Analysis by Using Capillary Zone Electrophoresis

“…Nowadays, CE has become a routine method for protein analyses in clinical, forensic and biomedical fields, as well as for the separation of proteins and peptides extracted from vegetable tissues [10]. Capillary zone electrophoresis (CZE) has been extensively applied to the analysis of wheat protein fractions (i.e., gliadins, glutenins and albumins) [11][12][13] for identifying grain variety by comparing the electrophoregrams [14], to investigate the variation of protein profiles within germplasm collections [15], to study the rate of protein accumulation in kernels [16,17], and so on. Despite this great versatility of CE in terms of efficiency and speed of analysis to investigate the different wheat protein fractions, no protocol exists for the investigation of the CM protein fraction.…”