Solvent-induced partial cellular fixation decodes proteome-wide drug targets and downstream pathways in living cells

Abstract Comprehensive knowledge of drug targets, their binding efficacy, and the subsequent activated cellular pathways is critical for determining therapeutic efficacy, elucidating mechanisms of action, and identifying potential side effects. However, acquiring this information in living cells remains challenging using routine proteomics methods. In this study, we propose a approach, the Solvent-Induced partial Cellular Fixation Approach (SICFA), to assess protein stability and drug interactions in living cells. By combining SICFA with quantitative proteomics, we can quantify stability for approximately 5,600 proteins within the intracellular proteome. SICFA demonstrates high sensitivity in detecting drug-induced stability changes in target proteins and downstream effector proteins. Time-resolved SICFA allows the tracking of drug-induced molecular events over time, revealing the early impact of 5-Fluorouracil on RNA post-transcriptional modifications and ribosome biogenesis. Unlike traditional proteomics, SICFA offers enhanced sensitivity in detecting early biochemical events, providing critical insights into drug-target interactions and the sequence of drug-induced pathway alterations.