Abstract 2439: Multiplexed chemoproteomic profiling as a tool to decipher the intracellular interactions between proteins and small molecules
Authors: Michael Ford, Richard Jones, Ravi Amunugama, Danette Daniels, Rachel Ohana, Thomas Kirkland, Marjeta Urh
Journal: Cancer Research (2015)

Abstract Chemoproteomic profiling is the qualitative and quantitative study of small molecule protein interactions using mass spectrometry. Chemoproteomics approaches typically involve four steps 1) modification of the bait drug molecules with the addition of an affinity tag, e.g. biotin, 2) incubation of the bait molecule with cells, cell lysates or tissue homogenates, 3) recovery of the bait molecule using the added affinity mechanism, e.g. streptavidin and 4) identification of proteins interacting with the bait molecule by liquid chromatography and tandem mass spectrometry (LC-MS/MS). To obtain value from chemoproteomics experiments any identified small molecule protein interactions need to be representative of the underlying biology and not artifacts of the chemoproteomics process. Chemoproteomic workflows offer a relatively simple method to generate a large volume of extremely valuable data about the behavior of a molecule, its analogs and its competitors. As such the number of samples generated for analysis can be significant. One method for maintaining throughput in chemoproteomics workflows is multiplexed proteomics with chemical labeling. This approach enables the pooling of multiple experimental conditions for analysis in a single LC-MS/MS experiment thus helping to reduce potential bottle necks associated with instrument capacity. Here we report a multiplexed chemoproteomics workflow based on a novel chloroalkane (CA) ligand that minimally affects compound potency and cell permeability using a p38 MAPKinase inhibitor. We show changes in protein interaction profiles by titrating cells with different mixtures of parent inhibitor + modified inhibitor-CA compound at varying relative concentrations. These changes in profiles can be monitored using cells transfected with NanoLuc fusion proteins of known inhibitor targets and show increasing capture correlated with increasing concentrations of modified inhibitor CA-compound. Overall interactions profiles can be more thoroughly analyzed by chemical labeling and combing this approach with 8plex iTRAQ labeling techniques. This multiplexing approach allows for many titrations points to be studies at once, yielding information about the dynamics of interactions between small molecules and their protein targets. The ability to study these differences after treatment of live cells aids in the understanding of the differing interaction targets of many given small molecule inhibitor. Citation Format: Michael Ford, Richard Jones, Ravi Amunugama, Danette Daniels, Rachel Ohana, Thomas Kirkland, Marjeta Urh. Multiplexed chemoproteomic profiling as a tool to decipher the intracellular interactions between proteins and small molecules. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2015-2439