Mass spectrometry-based proteomics is a powerful tool when combined with hypothesis driven protein purification. Regulation of protein-protein interactions is a major molecular mechanism for gene activation. Protein purification, functional assays, and antibody-based western blots have traditionally been used to elucidate many of the most critical and perhaps universal protein-protein interactions required for gene expression, such as the assembly of the general transcription machinery. The Mediator complex is an essential part of the general transcription machinery that integrates signals from DNA-binding transcriptional activators through protein-protein interactions to regulate RNA Pol II activity. Gene activation is ultimately determined by incoming stimuli and subsequent inter-cellular signaling. How these signals are integrated in a spatial and temporal fashion for the regulation of distinct genes by activator-Mediator interactions is unclear. One proposed molecular mechanism of gene activation by the Mediator complex is through a structural shift in the complex. Mediator structural shifts may trigger new protein-protein interactions required for transcription of select genes in response to a specific stimulus. To test this, Mediator complexes were purified with and without transcriptional activators. The activation domains of the transcriptional activators SREBP-1a and VP16, which generate distinct structures upon binding Mediator, were used to affinity purify activator-bound Mediator complexes. For comparison, antibodies for the Mediator subunits MED1 and CDK8 were used to affinity purify activator-free Mediator complexes. A mass spectrometry-based proteomics platform was established to characterize the protein compositions of each Mediator complex purification. The results showed additional cofactors in the activator-bound Mediator complexes, many of which had known function related to gene expression. Selected cofactors were validated for binding Mediator with an orthogonal purification that combined the activator and antibody purifications and western blotting. Together the proteomics data predicted and the western blotting confirmed new protein-protein interactions relevant for regulation of gene expression that were activator-specific. Collectively, these targeted proteomics approaches have generated many new hypotheses and have fundamentally altered our understanding of how gene expression is regulated
