Abstract

MS-based proteomics uniquely allows the direct and hypothesis-free analysis of small molecule interactions within the proteome. Chemoproteomics approaches classically rely on enrichment of drug targets from cell extracts or live cell systems by chemical probes derived from the bioactive compound under investigation. However, chemical probes can be challenging and time consuming to generate. Recently, several proteomics methods emerged that detect small molecule protein binding my measuring ligand-induced effects on biophysical properties of target proteins, e.g. thermal stability, resistance to proteolysis, and surface exposure of amino acid side chains. Combination of these methods with quantitative MS enable proteome-wide measurements. For example, thermal proteome profiling (TPP) or cellular thermal shift assay (CETSA)-MS deciphers a drug’s MoA by the hypothesis-free profiling of drug-induced protein thermal stability changes across the proteome. Whilst initial protocols only allowed detection of soluble intracellular targets; recent improvements demonstrated engagement of intra- and extracellular membrane proteins. The recently introduced cell surface thermal proteome profiling approach combines cell surface biotinylation with thermal shift assays and allows for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities. We present drug binding to extracellular receptors, complexes and transporters and describe stimulation-dependent remodeling of T-cell receptor complexes. In addition to cell surface proteins, protein secretion is an important factor with which cells can interact with their environment. Using a panel of liver toxic compounds, we demonstrate that the secretome of liver cell models allows clustering compounds with similar adverse mechanisms and when combined with thermal proteome profiling allows tracking dysregulated pathways and mechanism of action.