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 at high temperature, the counterpart, the insoluble
precipitate, has been overlooked for a long time. The analysis of
the 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 to the classic TPP method, but the process
of protein precipitation is assisted by microparticles. Upon heating,
proteins unfold and aggregate on the surface of the microparticles.
The introduction of a microparticle simplifies the whole sample preparation
workflow. The proteins that precipitate on the microparticles are
subjected to washing, alkylation, and digestion. The whole sample
preparation is processed conveniently on the surface of the microparticles
without any transfer. With the assistance of microparticles, sample
loss is minimized. The MAPS method is compatible with minute amounts
of initial proteins. MAPS was applied to screen the targets of several
well-studied drugs and the known target proteins were successfully
identified with high confidence and specificity. To investigate the
specificity of the method, MAPS was applied to screen the targets
of the pan-kinase inhibitor, staurosporine, and 32 protein kinases
(specificity of 80%) were identified using only 20 μg of initial
proteins of each sample. MAPS is an unbiased robust method for drug
target screening, filling the vacancy of stability-based target screening
using a precipitate.