Comprehensive profiling of CTP-binding proteins using a biotinylated CTP affinity probe

Abstract Recent studies have shown that CTP may act as a ligand to regulate the activity of its target proteins in many biological processes. However, proteome-wide identification of CTP-binding proteins remains challenging. Here, we employed a biotinylated CTP affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics approach to capture, identify and quantify CTP-binding proteins in human cells. By performing two types of competitive SILAC experiments with high vs. low concentrations of CTP probe (100 vs. 10 μmol/L) or with CTP probe in the presence of free CTP, we identified 90 potential CTP-binding proteins which are involved in a variety of biological processes, including protein folding, nucleotide binding and cell-cell adhesion. Together, we developed a chemical proteomic method for uncovering the CTP-binding proteins in human cells, which could be widely applicable for profiling CTP-binding proteins in other biological samples.