This work reports on the use of a three dimensional separation system to enhance metalloprotein information when considering soybean seeds. Separations using size exclusion chromatography (SEC) allowed identification of three metal fractions. Following an anion exchange (AEX) chromatographic separation in the second dimension, the resultant sub-fractions were lyophilized and subjected to a third dimension of separation using a polyacrylamide gel electrophoresis (SDS-PAGE). After the separation, the bands were digested, and, in addition to others, the following proteins, previously associated with metals, were identified: 3-lipoxygenase A chain (soybean) complex with 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid, beta-amylase [Glycine max], seed lipoxygenase-1, lipoxygenase [G. max], seed lipoxygenase-2 (Pisum sativum) and beta-conglycinin. Techniques presenting high resolution are desired due to their capability in resolving great amount of signals (responses) generated from hundreds of proteins generally found in different samples. To the best of our knowledge, this is the first time that bidimensional chromatographic system which allied to another separation dimension is applied for improving protein identification, so that higher number and different proteins were found when comparing 2D dimension with 3D dimension. In fact, this strategy is welcoming in proteomics studies, in order to improve the comprehension of those systems that present large number of proteins. This article is part of a Special Issue entitled: Environmental and structural proteomics.10494-103