3D Histology visualizing hypoxia-induced upregulation of N-terminal cysteine using de novo fluorophore generation.

Our research group developed a novel fluorescence staining strategy based on the DNFC targeting N-Cys in proteins. By treating biological samples with non-fluorogenic citrate and coupling reagents, we achieved strong cyan fluorescence, enabling effective visualization of N-Cys proteins in cells and tissues. The DNFC reaction occurs specifically on N-Cys residues, making it highly ideal for monitoring protein processing events, particularly within the Arg/N-degron pathway. Under hypoxic conditions, DNFC fluorescence is significantly enhanced, likely due to the increased presence of N-Cys-containing proteins. Using immunoassays and mass spectrometry, we identified Class 2 actin as a target protein under hypoxia, emphasizing the utility of 3D histopathology for analyzing actin's spatial distribution. Furthermore, we have identified a novel finding indicating a significant presence of RGS5 in red blood cells (RBCs), a discovery that has not been previously reported. Our fluorescence imaging studies, conducted across various cell types, animal tissues, and human clinical samples suggest that DNFC staining, when combined with tissue-clearing techniques, enables detailed 3D imaging of N-Cys proteins and may offer a means to assess molecular responses to hypoxia within tissues. This study highlights the potential of DNFC as a valuable tool for imaging and quantitative analysis of N-proteomes and providing a foundation for 3D histopathology in hypoxia-related disease research.