Abstract
Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.
Highlights
Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds
Abundant proteolytic peptide fragments between rapamycin and vehicle-treated samples were identified on a proteome-wide scale using a labelfree Data Independent Acquisition Mass Spectrometry (DIA-MS) approach[16]
LiPQuant substantially enriches for direct drug targets in both human cell lines and yeast with similar efficiency as other proteome-wide-scale techniques[6,21,37,38]
Summary
Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. These target capturing probes may perturb molecular interactions and biological functions Complementary strategies such as thermal proteome profiling (TPP)[6,7,8], stability of proteins from rates of oxidation (SPROX)[9] and drug affinity responsive target stability (DARTS)[10,11,12] map drug interactions by assessing variations of thermal stability, susceptibility to oxidation or to proteolytic degradation induced by ligand binding. We recently used limited proteolysis (LiP) to map metabolite binding proteins directly in whole cell lysates of microbial organisms (LiP-SMap) This is achieved through the global detection with liquid chromatography-coupled tandem mass spectrometry (LC-MS) of differential proteolytic patterns produced upon ligand binding[13]. We identify an unknown target of a research fungicide currently under study
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