Objective

The cellular thermal shift assay (CETSA) measures drug-targetengagement in cellular preparations or in tissues. The method provides realistic information about drug binding, drug transport and tissue distribution. The proximity extension assay (PEA) offers great sensitivity in analyses of large numbers of target proteins in small sample aliquots, or even in single cells. Here, we combined multiplex PEA assays in CETSA analyses and we compared the PEA results with mass spectrometry data for the same samples subjected to thermal shift. Model CETSA experiments were performed in human cancer cells, treated with the kinases inhibitors. PEA analyses were used to evaluatethermal shift assays in as little as 5000 cells. The CETSA-PEA results exhibited good correlation with those from MS, but MS required 200-fold more cells for analysis compared to PEA. This highly sensitive CETSA-PEA procedure can allow drug-target engagement to be monitored in small aliquots of patient material for analysis of drug binding in clinical settings.