Identification of copper-binding proteins in soybean seeds by immobilized metal affinity chromatography and mass spectrometry

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Metalloproteomics is defined as the structural and functional characterization of putative metal-binding proteins on a genome-wide scale. In this study, we carried out a systematic screen for copper-binding proteins in soybean seeds through the combined use of immobilized metal affinity chromatography and two-dimensional gel electrophoresis. A total of 32 protein spots displaying copper-binding ability were unambiguously identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis. About 78% of these identified proteins contain the possible copper-binding motifs, namely, H-(X)n-H (n = 0–5, 7, and 12), H-(X)3-C, H-(X)6-M, M-(X)7-H, and C-(X)n-C (n = 2–4). Available functional information suggested that the majority of the identified proteins are involved in storage, defense response, redox homeostasis, carbohydrate metabolism, and protein biosynthesis. Accordingly, the methodology reported here has the potential utility in additional metalloproteomic screening.