Highly Sensitive and Multiplexed Imaging of Protein Expression and Protein-Protein Interaction

The ability to profile each individual cell's biomolecules and molecular composition is crucial for understanding the underlying cellular heterogeneity of complicated biological systems. In the last decade, single-cell RNA sequencing has improved our knowledge of biology by allowing us to measure gene expressions at a high resolution with ease. Compared to genomic or transcriptomic assays, protein assays are much more naïve. Since the expression between mRNAs and proteins does not always correlate, measuring protein expressions adds value to current biological knowledge and provides different insights into the study. Here, a new approach for in-situ proteomic assay featuring cleavable fluorescence tyramide (CFT) and reiterative immunofluorescence was proposed and demonstrated to address the challenges of current proteomic technologies. The CFT-based proteomic assay possesses high multiplexity and sensitivity without the need for special instruments but standard fluorescence microscopy. A demonstration of the assay profiling thirty-nine proteins on a single FFPE tissue and subsequent data analysis was performed to support the potential of the approach. Lastly, an extended application of the CFT to characterize in-situ protein-protein interaction (PPI) was presented. With the ability to combine immunofluorescence and PPI characterization by applying CFT, the new molecule can be applied to various fields and bring proteomic technologies to the next level.