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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Publications at this Location » Publication #205747
Title: Extraction of Wheat Endosperm Proteins for Proteome Analysis

Author
item Hurkman Ii, William
item Tanaka, Charlene

Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/27/2006
Publication Date: 4/15/2007
Citation: Hurkman, W.J., Tanaka, C.K. 2007. Extraction of Wheat Endosperm Proteins for Proteome Analysis. Journal of Chromatography. 849: 344-350.

Interpretive Summary: Analysis of the protein composition of wheat endosperm is an important strategy in understanding and improving flour quality. High-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), the separation of proteins in the first dimension by charge and in the second dimension by size, is the method of choice for global protein identification. The key to high-resolution 2-DE is tailoring extraction and solubilization methods for optimal proteome coverage. Total protein extracts are valuable for analysis of the abundant gluten proteins, which have the unique elasticity and extensibility properties that determine flour functionality. However, protein fractionation is required to detect the many low abundance proteins also present in wheat endosperm. Sequential extraction takes advantage of the specific solubility properties of the different endosperm protein classes and effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. These fractionation approaches, when combined with 2-DE and protein identification, provide a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.

Technical Abstract: Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.