While the literature abounds with examples of metalloproteins that are involved in essential cellular processes, there are almost certainly numerous other proteins and peptides not yet identified which are involved in these morphogenic processes that bind metals such as copper, zinc, manganese and nickel. Existing technologies for the separation and identification of proteins from complex mixtures frequently rely upon denaturation of proteins. As the interaction of a protein with its metal ligand requires, minimally, the retention of three dimensional structure, we are developing approaches that allow us to maintain the native state of the protein. We are utilizing two separate methods for the isolation and identification of such metal-binding bioactive components. Immobilized metal affinity chromatography (IMAC) is being used to isolate proteins and peptides that specifically bind copper, zinc, manganese, or nickel from the extracellular milieu of our cell systems induced to undergo morphogenesis. The identity of these proteins will be deduced by mass spectrometry either of digested total recoverates, or by a combination of further separation followed by mass spectrometry of followed by algorithmic reconstruction of the initial data set. Such an approach should allow us to collect many of the metalloproteins involved in the regulation of morphogenesis. By comparing the profile of identified proteins over time during a morphogenic process, we will also be able to identify both differentially expressed metalloproteins, and newly formed bioactive metal-binding cryptic peptides resulting from digestion of existing extracellular matrix proteins. In a second approach, we will utilize pulse-chase labeling using radioactive metals (i.e. Cu-64 and Zn-75) followed by collection of extracellular proteins and native 2-D electrophoretic analysis and autoradiography. Alternatively, we will perform 2-D separation and transfer proteins to a solid matrix, refold the proteins, and allow them to interact with radioisotopic metals. Protein spots identified as putative metalloproteins by radioisotopic signature will be isolated and sequence identities determined by mass spectrometry. Such an approach should allow us to determine quantitative binding changes occurring during morphogenesis, as well as confirming the identity of newly formed cryptic bioactive peptides.