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Title: Determination of optimal protein quantity required to identify abundant and less abundant soybean seed proteins by 2D-PAGE and MS

item Natarajan, Savithiry - Savi
item Bae, Hanhong
item Caperna, Thomas
item Garrett, Wesley

Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/2/2007
Publication Date: 8/2/2007
Citation: Natarajan, S.S., Xu, C., Bae, H., Caperna, T.J., Garrett, W.M. 2007. Determination of optimal protein quantity required to identify abundant and less abundant soybean seed proteins by 2D-PAGE and MS. Plant Molecular Biology Reporter. 25:55-62.

Interpretive Summary: Genetically modified (GM) soybean is widely grown in the US and abroad and it is likely that soybean cultivars with a variety of modifications to enhance quality and productivity will be developed in the future. It is therefore important to determine if any unintended changes occur in the soybean seed as a result of genetic modification. We have standardized and applied a “proteomics” approach in which seed proteins are separated, and identified using a device called a “mass spectrometer” to determine the range of soybean seed proteins present in large amounts as well as those in lesser amounts. In this series of experiments we determined the quantities of total amounts of protein required to obtain optimal separation and identification of individual high abundance and low abundance soybean seed proteins. The data on protein seed composition of normal soybean are important in order to provide bases of comparison with transgenic soybeans. This comparison will allow scientists to identify unintended or collateral effects that may occur in the process of producing seed of new transgenic soybean cultivars. This will help to improve consumer acceptance of GM soybeans.

Technical Abstract: Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75 µg, 100 µg, 125 µg, 150 µg and 200 µg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry (MS) analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins (beta-conglycinin beta-homotrimer and glycinin G4 (A5A4B3) precursors) increased linearly between 75 and 125µg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 µg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer increased linearly with an increase in the amount of protein through 200 µg, whereas spot intensity of beta-conglycinin beta-homotrimer and the allergen Gly m bd 28K increased linearly until 150 µg and did not increase further at 200 ug. These results suggest that 150 µg protein was suitable amount for the separation of abundant proteins, and 200 µg protein was suitable for the separation of less abundant proteins prepared from soybean seeds.