Global analysis of endogenous protein disorder in cells

Disorder and flexibility in protein structures are essential for biological function but can also contribute to diseases, such as neurodegenerative disorders. However, characterizing protein folding on a proteome-wide scale within biological matrices remains challenging. Here we present a method using a bifunctional chemical probe, named TME, to capture in situ, enrich and quantify endogenous protein disorder in cells. TME exhibits a fluorescence turn-on effect upon selective conjugation with proteins with free cysteines in surface-exposed and flexible environments—a distinctive signature of protein disorder. Using an affinity-based proteomic approach, we identify both basal disordered proteins and those whose folding status changes under stress, with coverage to proteins even of low abundance. In lymphoblastoid cells from individuals with Parkinson’s disease and healthy controls, our TME-based strategy distinguishes the two groups more effectively than lysate profiling methods. High-throughput TME fluorescence and proteomics further reveal a universal cellular quality-control mechanism in which cells adapt to proteostatic stress by adopting aggregation-prone distributions and sequestering disordered proteins, as illustrated in Huntington’s disease cell models.