Microparticle assisted precipitation screening method for robust drug target identification

While thermal proteome profiling (TPP) shines in the field of drug target screening by analyzing the soluble fraction of the proteome samples treated with high temperature, the counterpart, insoluble precipitate has been overlooked for a long time. The analysis of precipitate is hampered by the inefficient sample processing procedure. Herein, we propose a novel method, termed Microparticle Assisted Precipitation Screening (MAPS), for drug target identification. The MAPS method exploits the principle that drug bound proteins will be more resistant to thermal unfolding similar as in classic TPP method, but the process of protein precipitation is assisted by microparticles. Upon heating, proteins will unfold and aggregate on the surface of microparticles, which benefits the following proteomic analysis of the precipitate. The introduction of microparticle simplifies the whole sample preparation workflow. The proteins that precipitate on the surface of microparticles will be subjected to wash, alkylation, and digestion. The whole sample preparation is processed conveniently on the surface of microparticles without any transferring. With the assistance of microparticles, sample loss is minimized. As a result, MAPS method is compatible with minute amount of initial proteins. MAPS was applied to screen the targets of several well-studied drugs and the known target proteins were successfully with high confidence and specificity. To investigate the specificity of MAPS method, it was applied it to screen the targets of the pan-kinase inhibitor, staurosporine, and 32 protein kinases (specificity of 80%) were identified by using only 20 µg initial proteins of each sample. MAPS is an unbiased robust method for identifying drug targets at the level of proteome, filling the vacancy of stability-based target screening using precipitate.