Abstract
BackgroundWheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences.ResultsGrain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%.ConclusionsThis is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression.
Highlights
Wheat flour is one of the world’s major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins
The major water-insoluble protein fraction, comprised largely of glutenin polymers and gliadin monomers, is often referred to as gluten; these proteins are categorized among the proline- and glutamine- rich cereal storage proteins known as prolamins
The utility of one of the major proteomics techniques, MS/MS, is limited for studies of flour proteins unless the following three conditions can be met: proteins must be cleaved into fragments suitable for MS/MS analysis, the sequence database must be adequate to discriminate among highly similar proteins, and the data analysis must account for the presence of highly similar proteins
Summary
Wheat flour is one of the world’s major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Variability in flour quality is a major problem for end users but the causes are poorly understood, partly because flour contains a complex mixture of similar but distinct proteins that are difficult to separate, identify and quantify [3]. The major water-insoluble protein fraction, comprised largely of glutenin polymers and gliadin monomers, is often referred to as gluten; these proteins are categorized among the proline- and glutamine- rich cereal storage proteins known as prolamins. High molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) are linked by disulfide bonds between Cys residues to form polymers that contribute strength and elasticity to flour doughs, whereas the monomeric gliadins contribute to dough viscosity and extensibility. Several early studies demonstrated the utility of 2-DE to visualize the total complement of flour proteins [21,22], and the combination of 2-DE and MS techniques offers great promise for identifying these many proteins [3,23,24]
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