2D-PELSA maps multidimensional target landscapes and reveals E2-GPX4 engagement associated with ferroptosis

Current methods for mapping ligand-protein interactions struggle to resolve weak yet critical binding events and conformational dynamics, particularly for pleiotropic endogenous hormones. Here, we develop a peptide-centric, dose-resolved strategy, the two-dimensional peptide-centric local stability assay (2D-PELSA), which enables highly reliable target identification and direct estimation of local binding affinities in native cellular environments. Dose-dependent information converts static proteolysis signatures into quantitative readouts of local structural remodeling at enhanced resolution, resolving direct engagement, allosteric regulation, complex remodeling, etc. Applied to 17β-estradiol (E2)-treated BT474 breast cancer cell lysates, 2D-PELSA identifies 1288 conformotypic E2-responsive peptides and 24 candidate targets enriched in unsaturated and long-chain fatty acid metabolism with micromolar EC50 values. GPX4 is identified as an E2-engaged ferroptosis regulator with an EC50 of 2.4 μM and is downregulated by micromolar E2. Orthogonal Western blot and MS-based assays validate this interaction in lysates and living cells. Integrated proteomic, lipidomic and phenotypic analyses show that chronic micromolar E2 exposure induces lipid metabolic reprogramming, lipid peroxidation and oxidative, iron-dependent cell death that is only partially rescued by ferrostatin-1 or deferoxamine. These findings establish 2D-PELSA as a versatile platform for dissecting ligand-induced structural responses and mapping target landscapes in complex biological contexts. Identifying ligand-binding proteins and determining their affinities is important but challenging in complex systems. Here, the authors develop 2D-PELSA, a peptide-centric, dose-resolved method for target identification and local affinity estimation and reveal an E2-GPX4 link to ferroptosis.