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2-D analysis of plant proteomes containing thousands of proteins has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the non-abundant proteins within seeds is difficult when 60-80% is storage proteins. Resolution can be improved through sample fractionation using separation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) to precipitate soybean (Glycine max) seed storage globulins, glycinin and beta-conglycinin. This method removes 87+/-4% of the highly abundant seed proteins from the extract, allowing for 541 previously inconspicuous proteins present in soybean seed to be more detectable (volume increase of >or=50%) using fluorescent detection. Of those 541 enhanced spots, 197 increased more than 2.5-fold when visualized with Coomassie. The majority of those spots were isolated and identified using peptide mass fingerprinting. Fractionation also provided detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2-D electrophoretic separation and an in-gel phosphoprotein stain. Application of this methodology toward other legumes, such as peanut, bean, pea, alfalfa and others, also containing high amounts of storage proteins, was examined, and is reported here.