Jiang, Y.; Movassaghi, C. S.; Munoz-Estrada, J.; Sundararaman, N.; Momenzadeh, A.; Meyer, J. G.

Large-scale mass spectrometry-based proteomic screening could reveal cellular mechanisms of drug action at systems resolution but remains limited by experimental complexity and the difficulty of extracting insight from high-dimensional datasets. Here, we describe an end-to-end platform that combines semi-automated sample preparation, rapid LC-MS/MS, and AI agent-based data analysis to enable scalable proteomic screening. In a screen of 172 compounds in HepG2 cells, we generated 1,232 proteomes with more than 8,700 quantified proteins in approximately three weeks. Agentic AI reduced data analysis and interpretation time to less than one day while translating proteomic measurements into structured mechanism-oriented summaries and experimentally testable hypotheses. Guided by this framework, we validated: (1) a cholesterol-lowering effect of methylene blue in vitro and (2) an association between loratadine exposure and increased circulating iron in matched electronic health record analyses. This work establishes a scalable platform for generating proteomic drug perturbation data and automatically converting that data into mechanistic insights and candidate translational hypotheses using AI.