Highly Sensitive Spatial Proteomics with Multicolor Cleavable Fluorescent Tyramide

High-resolution single-cell spatial proteomics offers transformative insights into cellular diversity, architecture, interactions, and functions within complex biological systems. However, the existing multiplexed protein imaging platforms face challenges such as limited detection sensitivity, constrained target multiplexing capacity, or technically demanding. To address these issues, we report a highly sensitive spatial proteomics approach, using multicolor cleavable fluorescent tyramide and off-the-shelf antibodies. This method employs horseradish peroxidase (HRP) to enzymatically deposit distinct fluorophores to stain varied target proteins. Through reiterative cycles of target labeling, fluorescence imaging, and fluorophore cleavage, this approach allows numerous proteins profiled at the optical resolution in the same specimen. Utilizing this technique, we quantified 38 proteins within a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest target multiplexing capacity achieved to date using tyramide signal amplification (TSA) methods. Analysis of ∼500,000 individual cells in the same tissue revealed distinct cell clusters based on their protein expression profiles and spatial microenvironment. By mapping the cells back to their original tissue locations, we observed specific tissue subregions are composed of unique cell clusters. Furthermore, we also studied the cell–cell interactions and found the cells from the same cluster often showed strong association, while the cells in the varied clusters usually avoided contact.