Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification.