A new method is described for identifying proteins participating in protein-protein interactions in a complex mixture. The protein mixture is partially reacted with cyanogen bromide-activated Sepharose such that 50% of the proteins are covalently bound. Proteins participating in noncovalent interactions with the bound proteins are retained through a washing step, then eluted and analyzed on a two-dimensional gel. Since the proteins are accessible to chemical manipulation, mass spectrometric identification of the spots can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extractfor proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, 8 were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Since native proteins are analyzed in their natural, native state, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.