Understanding protein structure is fundamental to address biological questions and diseases such as neurodegenerative disorders, diabetes and cancers. The diverse functions of proteins stem from their structural variability: some regions require complex folding to form well-defined three-dimensional structures necessary for their function, whereas others remain inherently disordered, enabling dynamic interactions with biomolecules. Remarkably, over half of the eukaryotic proteome is estimated to be intrinsically disordered or to contain disordered protein regions. However, studying these disordered proteins in their native, complex cellular environments on a proteome-wide scale remains challenging owing to the lack of high-throughput tools that enable analysis of such heterogeneous and dynamic proteins. These tools are essential to complement established methods such as X-ray crystallography, cryogenic electron microscopy, NMR and computational prediction methods.

To overcome current limitations, we developed an innovative, user-friendly fluorescent chemical probe, which we named TME after its chemical components. This cell-permeable probe enables the in situ capture, enrichment and quantification of disordered proteins or protein regions on a proteome-wide scale. TME fluorescence is activated upon selective binding to proteins with free cysteines located in surface-exposed and conformationally flexible environments — a hallmark of protein unfolding and disordered regions. We combined TME binding assays with mass spectrometry-based proteomics to introduce a workflow named reactive unfolded protein-based identification of cysteine on enrichment (RUBICON), used to enrich and quantify TME-labelled proteins. RUBICON effectively profiles basal and stress-induced disordered proteins across diverse organelles, with coverage of low-abundance proteins that are not detectable by traditional methods. We found that TME-labelled proteins are enriched in functional clusters including cell cycle, translation regulation, mRNA processing, nucleotide binding, quality control and biomolecular condensates, which were previously reported to be classified as intrinsically disordered proteins. Further validation confirmed that these proteins contain significantly disordered regions and act as hubs and bottlenecks in the protein–protein interaction network.