Comparison of co-immunoprecipitation techniques for effective identification of SNTA1 interacting proteins in breast cancer cells

Abstract

AbstractDiverse signal transduction pathways involve thousands of proteins acting in concert that contribute to breast cancer pathology. SNTA1 has emerged as a common link that functions to wire various complex signaling circuits within the cell. In our study we used endogenous SNTA1 protein as bait in pull-down assays to identify SNTA1 interactome from breast cancer cell extract. We compared the utility of two co-immunoprecipitation techniques conventional co-IP and cross-linking co-IP coupled to mass spectrometry (IP–MS) that intriguingly generated valuable inventory of SNTA1 associated proteins in breast cancer cells that may or may not interact physically but contribute to shared functions in breast cancer pathology. We observed that Pierce cross-link IP kit enhanced sensivity of the analysis as it decreased the number of background proteins with a remarkable increase in identified list of proteins. We have also observed that different approaches are able to consistently detect the same signaling outcome as observed by Gene Ontology. Identification of interacting partners of SNTA1 in breast cancer cell lines may depict the possible signaling mechanism of this protein in molecular pathogenesis of breast cancer and its possible use as a therapeutic target.