We have used the HaloTag™ to analyze the interactions of the human POLR2H protein, which is a protein shared between RNA Polymerase I, II, and III. Remarkably, use of this single subunit pulled down nearly all the major components of these three related, but distinct complexes.
We have developed a novel approach for assembling probabilistic local protein interaction networks using vector algebra and statistical methods, and applied this to the human Tip49a/Tip49b protein interaction network (Sardiu et al., 2008).
We utilize deletion network analyses to propose the architecture of multiprotein complexes and to determine the modularity of protein complexes. In our initial publication in this area, we analyzed the Rpd3/Sin3 histone deacetylase complexes.
After affinity purification to purify protein complexes from cells, we use the MudPIT and the dNSAF approach to quantitatively analyze multiprotein transcriptional regulatory complexes. The combination of these approaches provides not only a list of the proteins present but also the abundance of proteins present.
Our lab has worked with a number of external collaborators. We also work with nearly all the laboratories at the Stowers Institute to bring state of the art proteomics technologies to bear on biological problems.
Implementing the HaloTag™ For Protein Complex and Protein Network Analysis
The HaloTag™ is a multifunctional tag derived from a bacterial dehalogenase that forms covalent bonds with a variety of synthetic ligands (Promega, Madison WI). This system allows one to build a construct and cell lines that can be used in parallel for both affinity purification of protein complexes and imaging, for example. We are implementing the HaloTag™ as our primary choice for analysis of human protein complexes and protein interaction networks.
Quantitative Proteomic Analysis of Transcriptional Regulatory Complexes
The goal of our research is to develop label free quantitative proteomic tools with a particular focus on applying these tools to the analysis of transcriptional regulatory complexes. On the technology side, we are focused on the further development of spectral counting as a quantitative proteomic tool. On the biological side, we are particularly interested in the discovery of novel protein protein interactions with well characterized transcriptional regulatory complexes, the proteomic based analysis of the dynamics of transcriptional regulatory complexes, and characterization of protein interaction networks. We are a highly interactive and interdisciplinary group, and we collaborate extensively with many researchers at the Stowers Institute.
Dynamics of Multiprotein Complexes
Our research is focused on multiprotein complexes. Using affinity purification coupled with MudPIT, the NSAF, and now the dNSAF approach, we analyze complexes using different tagged subunits from the same multiprotein complex. This allows us to determine the relative abundance of particular proteins in a complex in a bait-dependent fashion and leads to the analysis of distinct forms of multiprotein complexes that can have important functional insights. In addition, current projects in the lab are using these approaches to determine the impact of different stimuli on multiprotein complexes.