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

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.

Elevated field atmospheric CO2 concentrations affect the characteristics of winter wheat (cv. Bologna) grains

“…Analysis of sequence coverage indicated that these spots were produced by cleavage of the N-termini of these proteins. These results are consistent with previous investigations indicating that globulin-3 family proteins can be processed at internal cleavage sites to yield polypeptides with a large range of molecular weights and pI values (Singh et al 2001;Dupont et al 2011;Koziol et al 2012). Interestingly, the spots corresponding to full-length globulin were downaccumulated under e[CO 2 ], whereas those corresponding to endo-proteolytic events accumulated or appeared in samples grown under these conditions.…”

A Multistrategic Approach in the Development of Sourdough Bread Targeted Towards Blood Pressure Reduction

“…Proteins in breads and sourdough were extracted following the procedure of Dupont et al [15] and separated under non- . Na and K were analysed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using a Perkin-Elmer Optima 7300-DV spectrophotometer (Waltham MA, USA), following microwave digestion of the samples with nitric acid and hydrogen peroxide.…”

Wheat Amylase Trypsin Inhibitors as Nutritional Activators of Innate Immunity

“…Finally, activity could be recovered in a non-gluten fraction that was present in standard gliadin and that was identified as the family of amylase trypsin inhibitors (ATIs) [15]. ATIs represents ∼2-4% of total wheat protein (as compared to ∼80-90% for gluten) [16]. …”