Affinity Purification-Mass Spectrometry
Protein-Protein Interactions (PPIs) play a vital role in cellular processes and fundamental biological insights can be gained by studying networks of interacting proteins rather than individual proteins. Also dysregulation and inappropriate activation of protein complexes are associated with several diseases, including cancer. In our Center, the Affinity Purification-Mass Spectrometry (AP-MS) platform is used to study the protein interactions that combine the specificity of antibody based protein purification with the sensitivity of mass spectrometry to identify putative interacting proteins. A recombinant FLAG-tagged bait of interest is first expressed in cultured cells, the bait protein and associated proteins are then retrieved using an antibody to the FLAG epitope, followed by identification of prey proteins using mass-spectrometry. Different scores such as Normal Spectral Abundance Factor (NSAF) and Z-score are used to prioritize the interacting proteins specific to the bait. For analyzing and visualizing the interaction network data, Spotfire platform and Cytoscape are used. Pathway Studio and Ingenuity Pathway Analysis (IPA) are also used to identify significant networks and for further annotation. AP-MS enables protein networks and complexes to be studied at the proteomic scale and it can be anticipated that the knowledge gathered from such projects will fuel drug target discovery and prove indispensable to the emerging field of systems biology.
Contacts:
Sudipto Saha, Ph.D. (sudipto.saha@case.edu)
Case Center for Proteomics and Bioinformatics
Case Western Reserve University
10900 Euclid Avenue, BRB 919
Cleveland, Ohio 44106
(phone) 216-368-2317
(fax) 216-368-6846