Submitted to: Analytical Biochemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/7/2005
Publication Date: 7/7/2005
Citation: Natarajan, S.S., Xu, C., Caperna, T.J., Garrett, W.M. 2005. Comparison of protein solubilization method suitable for proteomic analysis of soybean seed proteins. Analytical Biochemistry. 342: 214-220. Interpretive Summary: Soybean is the second most important cash crop in the U.S. with an estimated value of $15.2 billion. Genetically modified (GM) soybean is widely grown in the U.S. 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 important to determine if any unintended changes occur in the soybean seed as a result of genetic modification. Therefore, we have standardized and applied the technology to determine and quantify the spectrum of proteins present in soybean seed. To do this we are using a 'proteomics' approach in which seed proteins are separated, identified and quantified using a device called a mass spectrometer. This approach will be important in providing a way to identify any unintended or collateral effects that may be present in the seed of new transgenic soybean cultivars. This standardized methodology for defining and quantifying the spectrum of protein in soybean seed will be useful to scientists who wish to compare normal versus GM soybean.
Technical Abstract: Extraction of soybean seed proteins for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry analysis is challenging and inconsistent. In this study, we compared four different protein extraction/solubilization methods including urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone (TCA method) to determine their efficacy in separating soybean seed proteins by 2 D-PAGE. In all four methods, seed storage proteins were well separated by 2D- PAGE with minor variations in the intensity of the spots. The thiourea/urea and TCA methods showed higher protein resolution and spot intensity of all proteins compared with the other two methods. In addition, several less abundant and high molecular weight proteins were clearly resolved and strongly detected using the thiourea/urea and TCA methods. Protein spots obtained from the TCA method were subjected to mass spectrometry analysis to test their quality and compatibility. Fifteen protein spots were selected, digested with trypsin and were analyzed using Matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS). The proteins identified were ß-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, sucrose binding proteins, and seed maturation proteins. These results suggest that thiourea/urea and TCA methods are efficient and reliable methods for 2-D separation of soybean seed proteins and subsequent identification by mass spectrometry.