Development of a proteomic approach for identifying protein-protein interactions

• Main Author: Vasilescu, Julian
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/16209

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry. Transient protein complexes, which are characterized by high dissociation constants, are typically lost by this approach. The primary objective of this study is to describe a novel method for identifying transient protein-protein interactions using a combination of in vivo cross-linking, immunoaffinity purification, and mass spectrometry-based protein identification. Live cells expressing an epitope-tagged protein of interest are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to the protein of interest are co-purified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis, interacting proteins are identified by mass spectrometry. Application of this method enabled the identification of numerous proteins that co-purified with a constitutively active form of a Ras GTPase known as M-Ras (MRasQ71L), which was stably expressed in the murine mast cell line R6X. Among the proteins that were co-purified, the RasGAP-related protein IQGAP1 was shown to be a novel interaction partner of M-RasQ71L. We believe that this method is applicable to many proteins and will prove to be a valuable tool for the study of protein-protein interactions. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate