Antibody-array interaction mapping: a new method for studying protein-protein interactions and its application to the study of sera from pancreatic cancer patients

3832 Protein-protein interactions are major determinants of protein function, regulation and activity, and alterations in certain interactions can play roles in cancer pathology. A more thorough characterization of protein-protein interactions associated with disease could lead to improved diagnostic methods or treatment strategies. We present a new method for probing and measuring protein-protein interactions, called antibody-array interaction mapping (AAIM), which allows the efficient and sensitive measurement of interactions among a set of proteins in biological samples. A native, non-denatured biological sample, such as a serum sample, is incubated on replicate arrays of multiple antibodies, and after washing away unbound proteins, each array is probed with a different detection antibody, each antibody matched to one of the capture antibodies on the arrays. The binding patterns of the detection antibodies reveal interactions between the targets of the capture antibodies and the targets of the detection antibodies. The method is practical, rapid, and highly sensitive, and it can be used with native, biological samples, so that complex, multi-protein associations can be seen that would be missed with pairwise studies on purified proteins. Further, by using actual biological samples, one can view the real state of an interaction to assess its level in certain situations, which allows a better evaluation of the biological roles of interactions. The application of the method has revealed previously-uncharacterized interactions among cytokines, inflammatory mediators, and various glycoproteins in the sera of pancreatic cancer patients. Immuno-precipitation followed by western blot or mass spectrometry was used to validate two of the interactions, and others are currently under study. The measurement of these interactions in pools of sera from either pancreatic cancer patients or healthy control subjects shows that the levels of interactions between CRP and kininogen and between a CEA family member and IL-1beta may be associated with cancer. Since cytokine signaling and inflammatory processes play critical roles in processes that mediate cancer progression, the newly-observed interactions among inflammatory proteins could be involved in the regulation and control of these processes. Further, the measurement of the levels of certain interactions may prove valuable as a biomarker for certain disease states. Ongoing studies are aimed at further characterizing protein-protein interactions in serum and assessing the diagnostic potential and mechanistic implications of the interactions.