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United States Department of Agriculture

Agricultural Research Service

Research Project: Molecular Analysis of Effects of Environment on Wheat Flour Quality and Allergenic Potential

Location: Crop Improvement & Utilization Research

Title: Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

Authors
item Dupont, Frances
item Vensel, William
item Tanaka, Charlene
item Hurkman Ii, William
item Altenbach, Susan

Submitted to: Proteome Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 11, 2011
Publication Date: February 11, 2011
Citation: Dupont, F.M., Vensel, W.H., Tanaka, C.K., Hurkman II, W.J., Altenbach, S.B. 2011. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Science. 9(10) available:http://www.proteomesci.com/content/9/1/10.

Interpretive Summary: Wheat flour is one of the world's major food ingredients, but it is difficult to identify all the proteins in a flour sample. The abundant gluten proteins are responsible for many of the unique baking properties of wheat flour. They also create dietary problems for consumers with celiac disease or wheat allergies. However, it is challenging to tell all of these proteins apart and figure out which genes produce which proteins. This is the first successful effort to identify the majority of the individual flour proteins for a single wheat variety, relate them to their gene sequences and estimate their amounts. Use of a DNA database that included sequences from the variety of wheat that was used in the study was important. The study had to overcome many difficulties, including matching protein fragments with the correct gene sequences. This is particularly difficult because the gluten protein genes are very similar to each other and yet rapidly evolving. They are rich in the amino acids glutamine and proline and have very little arginine and lysine, which makes it more difficult to use proteases to break them into small pieces that can be identified using mass spectrometry. Prospects to simplify this process for routine analyses are discussed. Knowing which individual flour protein genes are turned into proteins and how abundant these proteins are will complement ongoing efforts efforts to sequence the wheat genome.

Technical Abstract: Wheat flour is one of the world's major food ingredients, but it is difficult to distinguish and identify the many proteins in a flour sample. The abundant glutamine and proline rich gluten proteins are responsible for many of the unique end-use qualities of wheat flour but it is challenging to distinguish them at the level of individual gene products. In this study, flour proteins were resolved and quantified by 2-DE and then identified by digestion with three proteases, followed by tandem mass spectrometry (MS/MS) analysis of the peptides, interrogation of the spectra using an improved protein sequence database, and use of the Scaffold program to integrate the results. Of the 373 most abundant spots, 232 were identified, accounting for 93% of total protein. High molecular weight glutenin subunits (HMW-GS) accounted for 17% of the flour protein, low molecular weight glutenin subunits (LMW-GS) 18%, alpha-gliadins 21%, gamma-gliadins 12%, omega-gliadins 10%, amylase-protease inhibitors 4%, "avenin-like" proteins 2%, triticins 1.5%, serpins 1.6%, beta-amylase 0.5%, other enzymes and factor 2.5% and globulins 0.4%. This is the first successful effort to identify the majority of the individual flour proteins for a single wheat variety, relate them to individual gene sequences and estimate their relative levels. Prospects to simplify this process for routine analyses are discussed.

Last Modified: 7/30/2014
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