U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska


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Published in Analytical Biochemistry 342 (2005) 214–220 doi:10.1016/j.ab.2005.04.046


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—urea, thiourea/urea, phenol, and a modified trichloroacetic acid (TCA)/acetone—to determine their efficacy in separating soybean seed proteins by 2D–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 analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) and liquid chromatography mass spectrometry (LC–MS). The proteins identified were β-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gly m Bd 28K allergen, and sucrose binding proteins. These results suggest that the thiourea/urea and TCA methods are efficient and reliable methods for 2D separation of soybean seed proteins and subsequent identification by mass spectrometry.