Technical Abstract: Large amounts of the major seed storage proteins, such as ß-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the less abundant proteins from soybean seeds in one dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10, 20, 30, 40, 50, 60, 70 and 80%) of isopropanol showed that greater than 30% isopropanal was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing above 40% isopropanol concentration resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using Matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches for lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.