Optimisation of protein extraction for in-depth profiling of the cereal grain proteome

Cereal grain proteomics can provide valuable information regarding plant growth, nutritional status, adaptation to environmental stresses, and the role that grain proteins play in health disorders, such as coeliac disease. In this study liquid chromatography-mass spectrometry was used to compare the barley proteome after extraction using seven protocols, with and without defatting and protein precipitation steps that aimed to remove interfering secondary metabolites. Tris-HCl and urea buffers yielded 1405 and 1483 proteins (~79% overlap) from barley (cv Sloop). Inclusion of a pre-extraction defatting step yielded 1336 (Tris-HCl) and 1286 (urea) proteins (~74% overlap). Whilst post-extraction TCA/acetone protein precipitation negatively impacted protein recovery, yielding 673 (Tris-HCl) and 734 (urea) proteins. Alcohol-based extraction yielded a lower number of proteins (645), but notably this extraction method co-extracted and enriched the gluten and α-amylase trypsin inhibitors. Based on these preliminary results, proteins were extracted from two selected cultivars of wheat, rye, barley and oats using three extraction protocols. Bioinformatic analyses of the identified proteins provide evidence that the choice of extraction buffer enriches different protein functional classes. The selection of the protein extraction protocol directly influences the identified cereal grain proteome composition, thus affecting the downstream biological interpretation of data. SIGNIFICANCE: LC-MS/MS and bioinformatics analysis revealed that both Tris-HCl and urea-based extraction yielded a similar suite of proteins from cereal grains with remarkable (70-80%) overlap. Yet the peptides derived from the proteins differed, rendering these extraction buffers complementary, in particular resulting in improved protein sequence coverage. The inclusion of commonly incorporated practices, such as pre-extraction defatting or post-extraction precipitation steps offered no benefit. The extraction method selected was noted to impact the downstream functional annotation results and biological interpretation.