Small molecule-protein interactions are critical to cell communication, nutrient scavenging, inhibition or activation of metabolic pathways, as enzyme cofactors, and numerous other processes. Small molecules are also used to control protein functions relevant to energy and human health, for instance to trigger a biosynthetic pathway that produces hydrocarbons or to inhibit a bacterial pathogen. However, discovery of the interactions occurring between small molecules and proteins in diverse biological systems is not simple. Activity-Based Protein Profiling (ABPP) is a chemical proteomic strategy that is often utilized to characterize enzyme function in native systems but is often a laborious process with isolation and purification of captured proteins. Thereby, a novel method for rapidly characterizing small molecule-protein interactions has significant potential to transform our understanding of biological functions. In our proposed assay, any arrangement and amount of chemical activity-based probes (drug/B vitamin mimics) are deposited into an array that is derivatized with probes that are immobilized to the surface of individual glass beads. Trypsin can then be directly added to the captured proteins, and the captured proteins measured by LC-MS. This will provide quantitative characterization of the proteins binding the drug or B vitamin (probe) molecules. Synthesis of a suite of probes will include orthogonal click chemistry (Azide, alkyne) partners and/or include a diazirine moiety for C-H insertion (covalent modification). The plate based assay can allow multiple different chemical probe compounds to be placed down to be screened against a single sample, or a plate could contain only a single chemical probe type but be rapidly screened against numerous sample types. The versatility of this array adds the utility of speed and tolerates small sample sizes to be screened in an efficient manner.

Glass surface adhered probe plate assay for characterization of protein binding partners of small molecules

254th National Meeting (2017)

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