|Hurkman Ii, William|
Submitted to: Journal of Cereal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2004
Publication Date: 11/1/2004
Citation: Hurkman, W.J., Tanaka, C.K. 2004. High resolution two-dimensional polyacrylamide gel electrophoresis of wheat endosperm proteins for proteome analysis. Journal of Cereal Science.40:295-299. Interpretive Summary: The proteins present in wheat flour are important determinants of bread baking quality, yet many of these proteins remain to be isolated and identified. High-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) is an invaluable tool for simultaneously separating and detecting the many hundreds of proteins present in flour. However, protein extraction and solubilization methods must be devised for optimal 2-D separations. In this paper, we evaluate several methods for analysis of total flour proteins and describe a fractionation procedure for isolating low abundance proteins that have not yet been identified. The methods reported here now make it possible to identify these low abundance proteins by mass spectrometry. The identifications will provide new information on biochemical processes in the wheat endosperm and their regulation. This information can in turn be applied to identifying gene products related to wheat yield and quality.
Technical Abstract: Successful proteomics studies of wheat endosperm proteins (Triticum aestivum L.) using two-dimensional gel electrophoresis (2-DE) and mass spectroscopy must rely on high-resolution gels, which in turn depend on protein fractionation and optimal solubilization methods. We evaluated protein extraction methods that utilized phenol, urea, SDS, or TCA. These methods all produced high-resolution 2-D gels with patterns dominated by the abundant gliadins and glutenins. To extend the analysis to low abundance proteins, flour proteins were fractionated into a KCl-insoluble, gliadin/glutenin fraction and KCl-soluble, albumin/globulin fraction. The 2-DE pattern of the KCl-insoluble fraction was similar to those of the total flour extracts. The albumin/globulin fraction was further fractionated into a methanol-soluble fraction that contained CM (chloroform-methanol soluble)-like proteins and a methanol-insoluble fraction substantially free of storage proteins. This multi-step fractionation method increased the number of flour proteins detected by 2-DE from approximately 250-300 in total protein extracts to nearly 900. The combination of the fractionation and high-resolution 2-DE methods reported here makes possible more detailed proteomics studies not only of the abundant storage proteins, but also of low abundance proteins involved in critical metabolic processes during grain-fill.