Orthogonal Chemical Proteomic Strategies Reveal the Cholesteryl Ester Interactome in Mammalian Cells

Cholesteryl esters (CEs) are widely regarded as inert storage forms of cholesterol, yet their potential to directly engage cellular proteins remains largely unexplored. Here, we establish an integrated chemical proteomic platform to systematically define CE-protein interactions in mammalian cells. We developed and comparatively deployed three orthogonal photoaffinity labeling strategies: (i) metabolic assembly of a bifunctional CE probe through endogenous acyl-CoA:cholesterol acyltransferase activity, (ii) direct delivery of a structurally defined diazirine-alkyne CE analog, and (iii) fragment-assisted subtraction using sterol and fatty acyl control probes to resolve interactions dependent on the intact esterified scaffold. Each probe system was rigorously validated by lipidomics and UV-dependent crosslinking prior to quantitative proteomic analysis. Integration of these complementary modalities identified 495 CE-associated proteins spanning enzymes, transporters, scaffolding proteins, and canonical sterol-binding families. The limited overlap across strategies reveals that lipid-protein engagement is strongly conditioned by biosynthetic origin, probe topology, and intracellular routing. Functional and database annotations further demonstrate enrichment of druggable and disease-linked proteins, connecting CE interactions to metabolic, neurological, and cardiovascular pathways. Collectively, this work provides the first systems-level map of CE-protein interactions and establishes a generalizable, multimodal framework for chemically resolving lipid-protein interactomes.