Associate Professor Harvard University, Massachusetts, United States
All biological processes are governed by chemical signals relayed through protein networks. These small molecule signals can inhibit, enhance, or impart new functions to proteins through direct associations to binding sites on a protein that drive alteration of the broader proteomic network. To discover binding site hotspots in the global proteome, we developed a chemical proteomics platform termed small molecule interactome mapping by photo-affinity labeling (SIM-PAL). SIM-PAL uses a small molecule carrying a photo-affinity label to capture molecular interactions within the global proteome. After treatment of live cells with the small molecule, the resulting interactions are captured by photochemical conjugation and affinity enriched. The enriched proteins are identified by proteomics and the exact binding sites are mapped by isotope-targeted mass spectrometry (MS). Isotope-targeted MS enables the selection of small molecule-linked peptides, representing binding sites, against a background of unlabeled peptides for high-confidence identification of the underlying molecular structure. SIM-PAL combines phenotypic cellular assays with high-resolution structural measurement of where and when a small molecule is binding throughout the whole proteome using the discovery power of MS. Applications of SIM-PAL to bioactive small molecules and the structural implications of binding site hotspots from photo-affinity labeling chemistry will be described.
