A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins

Abstract

Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of Brassica rapa R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE.

Shotgun proteomics of Brassica rapa seed proteins identifies vicilin as a major seed storage protein in the mature seed

“…Protein was extracted from seeds according to the method described in [ 41 ] with some modifications. In summary, seeds were placed in 2.0 mL PCR grade Safe Lock microcentrifuge tubes (Eppendorf AG, Germany, Lot: D15893R) with a single 5 mm stainless steel bead (Qiagen GmbH, D-40724, Hilden, Cat.…”

Identification, characterization and epitope mapping of proteins encoded by putative allergenic napin genes from Brassica rapa

“…To confirm the presence of napin proteins in Brassica rapa, a rapid, single seed extraction protocol was developed. 76 Single seeds were weighed and placed in 200 μL of 100% methanol in Safe Lock microcentrifuge tubes in the presence of a stainless-steel bead (5 mm, Qiagen GmbH, Hilden, Germany). The seed was ground using a TissueLyser (Qiagen, Australia) for two minutes, at 4°C and at the frequency of 20 oscillations per second.…”

Choosing the Right Path for the Successful Storage of Seeds

“…Consequently, there is a huge need for new biochemical or molecular markers of seed viability that improve the process of monitoring plant material during storage in gene banks and to help with the understanding of the concomitant aging process occurring in seeds. These studies have developed rapidly over the last decade and substantiate the importance of volatile organic compounds [ 94 , 95 , 98 ], proteomic studies [ 191 ], RNA integrity [ 192 , 193 , 194 , 195 , 196 ] and DNA damage markers or epigenetic marks [ 96 , 97 , 99 , 190 , 197 , 198 , 199 , 200 ] that are highly promising and should be more widely explored.…”