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

BACKGROUND: 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. 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. RESULTS: Grain 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%. CONCLUSIONS: This 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

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics

AbstractApplication of a thiol-specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin-linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed

An effective protein extraction method for two-dimensional electrophoresis in the anticancer herb Andrographis paniculata Nees

Proteomic analysis of plants relies on high yields of pure protein. In plants, protein extraction and purification present a great challenge due to accumulation of a large amount of interfering substances, including polysaccharides, polyphenols, and secondary metabolites. Therefore, it is necessary to modify the extraction protocols. A study was conducted to compare four protein extraction and precipitation methods for proteomic analysis. The results showed significant differences in protein content among the four methods. The chloroform-trichloroacetic acid-acetone method using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer provided the best results in terms of protein content, pellets, spot resolution, and intensity of unique spots detected. An overall of 83 qualitative or quantitative significant differential spots were found among the four methods. Based on the 2-DE gel map, the method is expected to benefit the development of high-level proteomic and biochemical studies of Andrographis paniculata, which may also be applied to other recalcitrant medicinal plant tissues

Evaluation of Biochemical and Quality Attributes to Monitor the Application of Heat and Cold Treatments in Tomato Fruit (Lycopersicon Esculentum Mill.)

The study was accomplished to optimize the application of stress treatment to prevent chilling injury (CI) in tomatoes. Four treatments were evaluated: control, heat shock applied for 24 (HS24) and 48 h (HS48), and cold shock applied for 24 h (CS24). The fruits were stored at 2 and 14C, and evaluated immediately after treatment and after 21 and 28 days of storage. An additional evaluation after 7 days of exposure at 20C following storage withdrawal was included to induce ripening and exteriorize the development of CI symptoms. Different biochemical and quality attributes were evaluated (color, titratable acidity, firmness, ethylene, CI index and heat shock protein [HSP] accumulation). Heat treatments were successful in preventing the decay of fruits under CI‐inducing conditions. The significantly better performance of HS48 in comparison to the other treatments gives evidence that the effectiveness relies on the adequate intensity of application; therefore, this intensity should be strictly controlled by a proper monitoring method. In this regard, small HSP accumulation was found suitable to reflect properly the physiological condition of fruits and therefore to potentially optimize and determine the adequate intensity of treatment application.Instituto de Tecnología de AlimentosFil: Polenta, Gustavo Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Budde, Claudio Olaf. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria San Pedro; ArgentinaFil: Sivakumar, Dharini. Tshwane University of Technology. Department of Crop Science. Postharvest Technology Group; SudáfricaFil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Guidi, Silvina Mabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentin