Proteomic analyses of protein complexes in the Wnt pathway

Multiple screens performed in Drosophila, Caenorhabditis elegans, Xenopus, and zebrafish have identified dozens of proteins participating in Wnt signal transduction. Epistasis experiments, enhancer and suppressor screens, and protein-protein interaction techniques have also been efficient at finding new pathway members, connecting proteins together, and establishing the architectural framework of how the Wnt signaling pathway functions. In the last few years, spectacular technological breakthroughs in the field of mass spectrometry have allowed the study of proteins and peptides with unprecedented sensitivity and accuracy. Recently, we have developed methods to study the Wnt pathway using mass spectrometry by studying the composition of protein complexes isolated from mammalian cells. In addition to identifying novel proteins acting in this pathway, this approach is providing information about the supramolecular organization of the protein complexes in the pathway and how the individual proteins are activated and regulated. This chapter details the experimental procedure that we developed to study mammalian protein complexes using a gel-free mass spectrometry approach.