Stability-Based Proteomic Methods Add Value to Activity-Based Protein Profiling Studies

inhibitor, ARS-1620, and the proteins in H358 cell lysates as a model system. Both methods identified ligand-induced protein folding stability shifts, collectively recovering the known on-target KRAS as well as multiple off-targets. Comparative analyses of the SPROX and TPP results with previously published pull-down data sets highlighted aldehyde dehydrogenase 1A3 (ALDH1A3) as a reproducible off-target that was missed in prior work using activity-based profiling methods. MS-based covalent site mapping identified C314 as the major ARS-1620 modification site on ALDH1A3, and enzymatic assays confirmed dose-dependent inhibition. Covalent docking supported a favorable binding pose within the retinal-binding pocket. Functionally, ALDH1A3 knockdown reduced ARS-1620-mediated cell killing, supporting the role of ALDH1A3 in ARS-1620's mode-of-action. Together, these findings not only demonstrate that stability-based proteomics provides a valuable and effective strategy for covalent drug target identification but also expand our knowledge of ARS-1620's biological activity.